Cyclic dinucleotides as sting agonists

ABSTRACT

Disclosed are compounds, compositions and methods for treating viral infections, diseases, syndromes, or disorders that are affected by the modulation of STING. Such compounds are represented by Formula (I) as follows:wherein R1A, R1B, R1c, B1, R2A, and R2B are defined herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Patent ApplicationNo. 62/426,350, filed Nov. 25, 2016; U.S. Provisional Patent ApplicationNo. 62/502,983, filed May 8, 2017; and U.S. Provisional PatentApplication No. 62/555,232, filed Sep. 7, 2017; which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to novel compounds which are STING(Stimulator of Interferon Genes) agonists and are useful for thetreatment of disorders that are affected by the modulation of the STINGprotein. The invention also relates to pharmaceutical compositionscomprising one or more of such compounds, processes to prepare suchcompounds and compositions, and use of such compounds or pharmaceuticalcompositions for the treatment of various diseases, syndromes anddisorders. The invention may be involved in the activation of thedownstream signaling pathway, further resulting in the activation ofsecond messengers and growth factors, and the production of interferoninvolved in the innate and adaptive immunity. More particularly, thepresent invention relates to the use of such compounds or pharmaceuticalcompositions for the treatment of various infections, diseases,syndromes and disorders including, but not limited to, melanoma, coloncancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma, andantiviral therapy.

BACKGROUND OF THE INVENTION

STING (stimulator of interferon genes), also known as TMEM173, MITA,MPYS, and EMS, is a transmembrane receptor located inside the cell and akey sensor of cytosolic nucleic acids (Zhong B, et al. “The AdaptorProtein MITA Links Virus-Sensing Receptors to IRF3 Transcription FactorActivation”. Immunity. 2008. vol. 29: 538-550). Recent studies haverevealed the biology of STING and its role in mobilizing an innateimmune response resulting in robust antitumor activity in mouse models.Activation of the STING pathway results in production of Type Iinterferons (mainly IFN-α and IFN-β) induced through the IRF3(interferon regulatory factor 3) pathway. Activation of IRF3 is thoughtto be mediated by TBK1 that recruits and phosphorylates IRF3 thusforming an IRF3 homodimer capable of entering the nucleus to transcribetype I interferon and other genes (Liu S, et al. “Phosphorylation ofinnate immune adaptor proteins MAVS, STING, and TRIF induces IRF3activation” Science. 2015: 2630-2637). TBK1 also activates the nuclearfactor kappa-light-chain-enhancer of activated B cells pathway whichleads to production of pro-inflammatory cytokines (IL-1α, IL-1β, IL-2,IL-6, TNF-α, etc.), via the oncogenic transcription factor NF-_(K)B. Inaddition, STING activates STAT6 (signal transducer and activator oftranscription 6) to induce (Th2-type), increase (IL-12) or decrease(IL-10) production of various cytokines, including the chemokines CCL2,CCL20, and CCL26 (Chen H, et al. “Activation of STAT6 by STING IsCritical for Antiviral Innate Immunity” Cell. 2011, vol. 14: 433-446).Direct phosphorylation of STING on Ser366 upon activation has also beenreported to occur through TBK1 (Corrales, L. et al “Direct activation ofSTING in the tumor microenvironment leads to potent and systemic tumorregression and immunity” Cell Reports, 2015, vol. 11: 1-13; Konno, H. etal. “Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STINGto prevent sustained innate immune signaling” Cell, 2013, vol. 155:688-698).

The natural ligand that binds to and activates STING (2′,3′)cyclicguanosine monophosphate-adenosine monophosphate (2′,3′-cGAMP) and theenzyme responsible for its synthesis (cGAS, also known as C6orf150 orMB21D1) have been elucidated providing an opportunity to modulate thispathway. cGAMP is a high affinity ligand for STING produced in mammaliancells that serves as an endogenous second messenger to activate theSTING pathway. It is a cyclic dinucleotide with a unique 2′, 3′ linkageproduced by cGAS in the presence of exogenous double-stranded DNA (e.g.that released by invading bacteria, viruses or protozoa) or of self-DNAin mammals (Wu et al., 2013; Sun, L. et al. “Cyclic GMP-AMP Synthase Isa Cytosolic DNA Sensor That Activates the Type I Interferon Pathway”Science, 2013, vol. 339: 786-791; Bhat N and Fitzgerald K A.“Recognition of Cytosolic DNA by cGAS and other STING-dependentsensors”. Eur J Immunol. 2014 March; 44(3):634-40). STING activation canalso occur through binding of exogenous (3′,3) cyclic dinucleotides(c-di-GMP, c-di-AMP and 3′3′-cGAMP) that are released by invadingbacteria (Zhang X, et al. “Cyclic GMP-AMP Containing MixedPhosphodiester Linkages Is An Endogenous High-Affinity Ligand for STING”Molecular Cell, 2013, vol. 51: 226-235; Danilchanka, O and Mekalanos, JJ. “Cyclic Dinucleotides and the Innate Immune Response” Cell. 2013.vol. 154: 962-970).

Activation of the STING pathway triggers an immune response that resultsin generation of specific killer T-cells that can shrink tumors andprovide long lasting immunity so they do not recur. The strikingantitumor activity obtained with STING agonists in preclinical modelshas generated a high level of excitement for this target and smallmolecule compounds that can modulate the STING pathway have potential totreat both cancer and reduce autoimmune diseases.

Activation of the STING pathway also contributes to an antiviralresponse. Loss-of-functional response, either at the cellular ororganism level, demonstrates an inability to control viral load in theabsence of STING. Activation of the STING pathway triggers an immuneresponse that results in antiviral and proinflammatory cytokines thatcombat the virus and mobilize the innate and adaptive arms of the immunesystem. Ultimately, long-lasting immunity is developed against thepathogenic virus. The striking antiviral activity obtained with STINGagonists in preclinical models has generated a high level of excitementfor this target and small molecule compounds that can modulate the STINGpathway have potential to treat chronic viral infections, such ashepatitis B.

Chronic hepatitis B virus (HBV) infection is a significant global healthproblem, affecting over 5% of the world population (over 350 millionpeople worldwide and 1.25 million individuals in the U.S.). Despite theavailability of certain HBV vaccines and therapies, the burden ofchronic HBV infection continues to be a significant unmet worldwidemedical problem due to suboptimal treatment options and sustained ratesof new infections in most parts of the developing world. Currenttreatments are limited to only two classes of agents: interferon alphaand nucleoside analogues acting as inhibitors of the viral polymerase.Yet none of these therapies offer a cure to the disease, and drugresistance, low efficacy, and tolerability issues limit their impact.The low cure rates of HBV are attributed at least in part to the factthat complete suppression of virus production is difficult to achievewith a single antiviral agent. However, persistent suppression of HBVDNA slows liver disease progression and helps to prevent hepatocellularcarcinoma. Current therapy goals for HBV-infected patients are directedto reducing serum HBV DNA to low or undetectable levels, and toultimately reducing or preventing the development of cirrhosis andhepatocellular carcinoma. There is, therefore, a need in the art fortherapeutic agents that can increase the suppression of virus productionand that can treat, ameliorate, or prevent HBV infection. Administrationof such therapeutic agents to an HBV infected patient, either asmonotherapy or in combination with other HBV treatments or ancillarytreatments, may lead to significantly reduced virus burden, improvedprognosis, diminished progression of the disease and enhancedseroconversion rates.

The potential therapeutic benefits of enhancing both innate and adaptiveimmunity make STING an attractive therapeutic target that demonstratesimpressive activity by itself and can also be combined with otherimmunotherapies.

SUMMARY OF THE INVENTION

The present invention is directed to compounds of Formula (I)

wherein

R_(1A) is hydroxy or fluoro and R_(1C) is hydrogen; or, R_(1A) is —O—and R_(1C) is CH₂ such that R_(1A) and R_(1C) are taken together withthe atoms to which they are attached to form a 5-membered ring;

R_(1B) is selected from the group consisting of hydroxy, thiol, and BH₃⁻;

B₁ is selected from the group consisting of rings b1 and b2

-   -   R_(2A) is selected from the group consisting of hydroxy and        methoxy;

R_(2B) is selected from the group consisting of hydroxy, thiol, and BH₃⁻;

provided that the compound of Formula (I) is other than(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-Amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-3,12,18-trihydroxy-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0⁶,¹⁰]octadecane-3,12-dione, bis-ammoniumsalt;

or an enantiomer, diastereomer, or pharmaceutically acceptable salt formthereof.

The present invention also provides a pharmaceutical compositioncomprising, consisting of and/or consisting essentially of apharmaceutically acceptable carrier, a pharmaceutically acceptableexcipient, and/or a pharmaceutically acceptable diluent and a compoundof Formula (I), or a pharmaceutically acceptable salt form thereof.

Also provided are processes for making a pharmaceutical compositioncomprising, consisting of, and/or consisting essentially of admixing acompound of Formula (I), and a pharmaceutically acceptable carrier, apharmaceutically acceptable excipient, and/or a pharmaceuticallyacceptable diluent.

The present invention further provides methods for treating orameliorating a viral infection, disease, syndrome, or condition in asubject, including a mammal and/or human in which the viral infection,disease, syndrome, or condition is affected by the agonism of STING,using a compound of Formula (I).

The present invention further provides methods for treating orameliorating a viral infection, disease, syndrome, or condition in asubject, including a mammal and/or human, using a compound of Formula(I).

The present invention further provides methods for treating orameliorating a viral infection, disease, syndrome, or condition in asubject, including a mammal and/or human in which the viral infection,disease, syndrome, or condition is affected by the agonism of STING,selected from the group consisting of melanoma, colon cancer, breastcancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B,using a compound of Formula (I).

The present invention further provides methods for treating orameliorating a viral infection, disease, syndrome, or condition in asubject, including a mammal and/or human, selected from the groupconsisting of melanoma, colon cancer, breast cancer, prostate cancer,lung cancer, fibrosarcoma, and hepatitis B, using a compound of Formula(I).

The present invention is also directed to the use of any of thecompounds described herein in the preparation of a medicament whereinthe medicament is prepared for treating a viral infection, disease,syndrome, or condition that is affected by the agonism of STING,selected from the group consisting of melanoma, colon cancer, breastcancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B, ina subject in need thereof.

The present invention is also directed to the use of any of thecompounds described herein in the preparation of a medicament whereinthe medicament is prepared for treating a viral infection, disease,syndrome, or condition selected from the group consisting of melanoma,colon cancer, breast cancer, prostate cancer, lung cancer, fibrosarcoma,and hepatitis B, in a subject in need thereof.

The present invention is also directed to the preparation of substitutedcyclic dinucleotide derivatives that act as selective agonists of STING.

Exemplifying the invention are methods of treating a viral infection,disease, syndrome, or condition modulated by STING selected from thegroup consisting of melanoma, colon cancer, breast cancer, prostatecancer, lung cancer, fibrosarcoma, and hepatitis B, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of any of the compounds or pharmaceutical compositions describedabove.

Exemplifying the invention are methods of treating a viral infection,disease, syndrome, or condition selected from the group consisting ofmelanoma, colon cancer, breast cancer, prostate cancer, lung cancer,fibrosarcoma, and hepatitis B, comprising administering to a subject inneed thereof a therapeutically effective amount of any of the compoundsor pharmaceutical compositions described above.

In another embodiment, the present invention is directed to a compoundof Formula (I) for use in the treatment of a viral infection, disease,syndrome, or condition affected by the agonism of STING selected fromthe group consisting of melanoma, colon cancer, breast cancer, prostatecancer, lung cancer, fibrosarcoma, and hepatitis B.

In another embodiment, the present invention is directed to acomposition comprising a compound of Formula (I) for the treatment of aviral infection, disease, syndrome, or condition selected from the groupconsisting of melanoma, colon cancer, breast cancer, prostate cancer,lung cancer, fibrosarcoma, and hepatitis B.

DETAILED DESCRIPTION OF THE INVENTION

With reference to substituents, the term “independently” refers to thesituation where when more than one substituent is possible, thesubstituents may be the same or different from each other.

The term “alkyl” whether used alone or as part of a substituent group,refers to straight and branched carbon chains having 1 to 8 carbonatoms. Therefore, designated numbers of carbon atoms (e.g., C₁₋₈) referindependently to the number of carbon atoms in an alkyl moiety or to thealkyl portion of a larger alkyl-containing substituent. In substituentgroups with multiple alkyl groups such as, (C₁₋₆alkyl)₂amino-, theC₁₋₆alkyl groups of the dialkylamino may be the same or different.

The term “alkoxy” refers to an —O-alkyl group, wherein the term “alkyl”is as defined above.

The terms “alkenyl” and “alkynyl” refer to straight and branched carbonchains having 2 to 8 carbon atoms, wherein an alkenyl chain contains atleast one double bond and an alkynyl chain contains at least one triplebond.

The term “cycloalkyl” refers to saturated or partially saturated,monocyclic or polycyclic hydrocarbon rings of 3 to 14 carbon atoms.Examples of such rings include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and adamantyl.

The term “heterocyclyl” refers to a nonaromatic monocyclic or bicyclicring system having 3 to 10 ring members that include at least 1 carbonatom and from 1 to 4 heteroatoms independently selected from N, O, andS. Included within the term heterocyclyl is a nonaromatic cyclic ring of5 to 7 members in which 1 to 2 members are N, or a nonaromatic cyclicring of 5 to 7 members in which 0, 1 or 2 members are N and up to 2members are O or S and at least one member must be either N, O, or S;wherein, optionally, the ring contains 0 to 1 unsaturated bonds, and,optionally, when the ring is of 6 or 7 members, it contains up to 2unsaturated bonds. The carbon atom ring members that form a heterocyclering may be fully saturated or partially saturated.

The term “heterocyclyl” also includes two 5 membered monocyclicheterocycloalkyl groups bridged to form a bicyclic ring. Such groups arenot considered to be fully aromatic and are not referred to asheteroaryl groups. When a heterocycle is bicyclic, both rings of theheterocycle are non-aromatic and at least one of the rings contains aheteroatom ring member. Examples of heterocycle groups include, and arenot limited to, pyrrolinyl (including 2H-pyrrole, 2-pyrrolinyl or3-pyrrolinyl), pyrrolidinyl, imidazolinyl, imidazolidinyl, pyrazolinyl,pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, andpiperazinyl. Unless otherwise noted, the heterocycle is attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure.

The term “aryl” refers to an unsaturated, aromatic monocyclic orbicyclic carbocyclic ring of 6 to 10 carbon members. Examples of arylrings include phenyl and naphthalenyl.

The term “heteroaryl” refers to an aromatic monocyclic or bicyclic ringsystem having 5 to 10 ring members, which contains carbon atoms and from1 to 4 heteroatoms independently selected from the group consisting ofN, O, and S. Included within the term heteroaryl are aromatic rings of 5or 6 members wherein the ring consists of carbon atoms and has at leastone heteroatom member. Suitable heteroatoms include nitrogen, oxygen,and sulfur. In the case of 5 membered rings, the heteroaryl ringpreferably contains one member of nitrogen, oxygen or sulfur and, inaddition, up to 3 additional nitrogens. In the case of 6 membered rings,the heteroaryl ring preferably contains from 1 to 3 nitrogen atoms. Forthe case wherein the 6 membered ring has 3 nitrogens, at most 2 nitrogenatoms are adjacent. Examples of heteroaryl groups include furyl,thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl,isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl,pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, isoindolyl,benzofuryl, benzothienyl, indazolyl, benzimidazolyl, benzothiazolyl,benzoxazolyl, benzisoxazolyl, benzothiadiazolyl, benzotriazolyl,quinolinyl, isoquinolinyl and quinazolinyl. Unless otherwise noted, theheteroaryl is attached to its pendant group at any heteroatom or carbonatom that results in a stable structure.

The term “halogen” or “halo” refers to fluorine, chlorine, bromine andiodine atoms.

Whenever the term “alkyl” or “aryl” or either of their prefix rootsappear in a name of a substituent (e.g., arylalkyl, alkylamino) the nameis to be interpreted as including those limitations given above for“alkyl” and “aryl.” Designated numbers of carbon atoms (e.g., C₁-C₆)refer independently to the number of carbon atoms in an alkyl moiety, anaryl moiety, or in the alkyl portion of a larger substituent in whichalkyl appears as its prefix root. For alkyl and alkoxy substituents, thedesignated number of carbon atoms includes all of the independentmembers included within a given range specified. For example, C₁₋₆alkylwould include methyl, ethyl, propyl, butyl, pentyl and hexylindividually as well as sub-combinations thereof (e.g., C₁₋₂, C₁₋₃,C₁₋₄, C₁₋₅, C₂₋₆, C₃₋₆, C₄₋₆, C₅₋₆, C₂₋₅, etc.).

In general, under standard nomenclature rules used throughout thisdisclosure, the terminal portion of the designated side chain isdescribed first followed by the adjacent functionality toward the pointof attachment. Thus, for example, a “C₁-C₆ alkylcarbonyl” substituentrefers to a group of the formula:

The term “R” at a stereocenter designates that the stereocenter ispurely of the R-configuration as defined in the art; likewise, the term“S” means that the stereocenter is purely of the S-configuration. Asused herein, the terms “*R” or “*S” at a stereocenter are used todesignate that the stereocenter is of pure but unknown configuration. Asused herein, the term “RS” refers to a stereocenter that exists as amixture of the R- and S-configurations. Similarly, the terms “*RS” or“*SR” refer to a stereocenter that exists as a mixture of the R- andS-configurations and is of unknown configuration relative to anotherstereocenter within the molecule.

Compounds containing one stereocenter drawn without a stereo bonddesignation are a mixture of two enantiomers. Compounds containing twostereocenters both drawn without stereo bond designations are a mixtureof four diastereomers. Compounds with two stereocenters both labeled“RS” and drawn with stereo bond designations are a two-component mixturewith relative stereochemistry as drawn. Compounds with two stereocentersboth labeled “*RS” and drawn with stereo bond designations are atwo-component mixture with relative stereochemistry unknown. Unlabeledstereocenters drawn without stereo bond designations are a mixture ofthe R- and S-configurations. For unlabeled stereocenters drawn withstereo bond designations, the absolute stereochemistry is as depicted.

Unless otherwise noted, it is intended that the definition of any substituent or variable at a particular location in a molecule beindependent of its definitions elsewhere in that molecule. It isunderstood that substituents and substitution patterns on the compoundsof the present invention can be selected by one of ordinary skill in theart to provide compounds that are chemically stable and that can bereadily synthesized by techniques known in the art as well as thosemethods set forth herein.

The term “subject” refers to an animal, preferably a mammal, mostpreferably a human, who has been the object of treatment, observation orexperiment.

The term “therapeutically effective amount” refers to an amount of anactive compound or pharmaceutical agent, including a compound of thepresent invention, which elicits the biological or medicinal response ina tissue system, animal or human that is being sought by a researcher,veterinarian, medical doctor or other clinician, which includesalleviation or partial alleviation of the symptoms of the disease,syndrome, condition, or disorder being treated.

The term “composition” refers to a product that includes the specifiedingredients in therapeutically effective amounts, as well as any productthat results, directly, or indirectly, from combinations of thespecified ingredients in the specified amounts.

The term “STING agonist” is intended to encompass a compound thatinteracts with STING by binding to it and inducing downstream signaltransduction characterized by activation of the molecules associatedwith STING function. This includes direct phosphorylation of STING, IRF3and/or NF-_(K)B and could also include STAT6. STING pathway activationresults in increased production of type I interferons (mainly IFN-α andIFN-β) and expression of interferon-stimulated genes (Chen H, et al.“Activation of STAT6 by STING Is Critical for Antiviral InnateImmunity”. Cell. 2011, vol. 14: 433-446; and Liu S-Y, et al. “Systematicidentification of type I and type II interferon-induced antiviralfactors”. Proc. Natl. Acad. Sci. 2012:vol. 109 4239-4244).

The term “STING-modulated” is used to refer to a condition affected bySTING directly or via the STING pathway, including but not limited to,viral infections, diseases or conditions such as melanoma, colon cancer,breast cancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitisB infection.

As used herein, unless otherwise noted, the term “disorder modulated bySTING” shall mean any viral infection, disease, disorder or conditioncharacterized in that at least one of its characteristic symptoms isalleviated or eliminated upon treatment with a STING agonist. Suitableexamples include, but are not limited to melanoma, colon cancer, breastcancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B.

As used herein, unless otherwise noted, the term “affect” or “affected”(when referring to a viral infection, disease, syndrome, condition ordisorder that is affected by agonism of STING) includes a reduction inthe frequency and/or severity of one or more symptoms or manifestationsof said viral infection, disease, syndrome, condition or disorder;and/or include the prevention of the development of one or more symptomsor manifestations of said viral infection, disease, syndrome, conditionor disorder or the development of the viral infection, disease,condition, syndrome or disorder.

The compounds of the instant invention are useful in methods fortreating or ameliorating a viral infection, disease, a syndrome, acondition or a disorder that is affected by the agonism of STING. Suchmethods comprise, consist of and/or consist essentially of administeringto a subject, including an animal, a mammal, and a human in need of suchtreatment, amelioration and/or prevention, a therapeutically effectiveamount of a compound of Formula (I), or an enantiomer, diastereomer,solvate or pharmaceutically acceptable salt thereof.

In particular, the compounds of Formula (I), or an enantiomer,diastereomer, solvate or pharmaceutically acceptable salt form thereofare useful for treating or ameliorating diseases, syndromes, conditions,or disorders such as melanoma, colon cancer, breast cancer, prostatecancer, lung cancer, fibrosarcoma, and hepatitis B.

More particularly, the compounds of Formula (I), or an enantiomer,diastereomer, solvate or pharmaceutically acceptable salt form thereofare useful for treating or ameliorating melanoma, colon cancer, breastcancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a compound of Formula (I), or an enantiomer,diastereomer, solvate or pharmaceutically acceptable salt form thereofas herein defined.

Some embodiments disclosed herein relate to methods of amelioratingand/or treating a viral infection including infections caused byHepadnaviridae such as hepatitis B virus or HBV. The methods can includeadministering to a subject identified as suffering from a viralinfection an effective amount of one or more compounds of Formula (I),or a pharmaceutically acceptable salt form thereof, or a pharmaceuticalcomposition that includes one or more compounds of Formula (I), or apharmaceutically acceptable salt form thereof.

Other embodiments disclosed herein relate to a method of amelioratingand/or treating a viral infection that can include contacting a cellinfected with the virus with an effective amount of one or morecompounds described herein (for example, a compound of Formula (I), or apharmaceutically acceptable salt form thereof), or a pharmaceuticalcomposition that includes one or more compounds described herein, or apharmaceutically acceptable salt thereof. Still other embodimentsdescribed herein relate to using one or more compounds of Formula (I),or a pharmaceutically acceptable salt form thereof, in the manufactureof a medicament for ameliorating and/or treating a viral infection.

Yet still other embodiments described herein relate to one or morecompounds of Formula (I), or a pharmaceutically acceptable salt formthereof, or a pharmaceutical composition that includes one or morecompounds of Formula (I), or a pharmaceutically acceptable salt formthereof, that can be used for ameliorating and/or treating a viralinfection. Some embodiments disclosed herein relate to a method ofinhibiting replication of a virus that can include contacting a cellinfected with the virus with an effective amount of one or morecompounds of Formula (I), or a pharmaceutically acceptable salt formthereof, or a pharmaceutical composition that includes one or morecompounds described herein, or a pharmaceutically acceptable salt formthereof.

Other embodiments described herein relate to using one or more compoundsof Formula (I), or a pharmaceutically acceptable salt form thereof) inthe manufacture of a medicament for inhibiting replication of a virus.Still other embodiments described herein relate to one or more compoundsdescribed herein (for example, a compound of Formula (I), or apharmaceutically acceptable salt form thereof), or a pharmaceuticalcomposition that includes one or more compounds described herein, or apharmaceutically acceptable salt form thereof, that can be used forinhibiting replication of a virus.

In some embodiments, the viral infection can be a hepatitis B viralinfection. The methods can include administering to a subject identifiedas suffering from HBV an effective amount of one or more compounds ofFormula (I), or a pharmaceutically acceptable salt form thereof, or apharmaceutical composition that includes one or more compounds ofFormula (I), or a pharmaceutically acceptable salt form thereof.

Other embodiments disclosed herein relate to a method of amelioratingand/or treating a viral infection that can include contacting a cellinfected with HBV with an effective amount of one or more compounds ofFormula (I), or a pharmaceutically acceptable salt form thereof, or apharmaceutical composition that includes one or more compounds ofFormula (I), or a pharmaceutically acceptable salt form thereof. Stillother embodiments described herein relate to using one or more compoundsof Formula (I), or a pharmaceutically acceptable salt form thereof, inthe manufacture of a medicament for ameliorating and/or treating HBV.

Yet still other embodiments described herein relate to one or morecompounds of Formula (I), or a pharmaceutically acceptable salt formthereof, or a pharmaceutical composition that includes one or morecompounds of Formula (I), or a pharmaceutically acceptable salt formthereof, that can be used for ameliorating and/or treating HBV. Someembodiments disclosed herein relate to a method of inhibitingreplication of HBV that can include contacting a cell infected with thevirus with an effective amount of one or more compounds of Formula (I),or a pharmaceutically acceptable salt form thereof, or a pharmaceuticalcomposition that includes one or more compounds of Formula (I), or apharmaceutically acceptable salt thereof.

Other embodiments described herein relate to using one or more compoundsof Formula (I), or a pharmaceutically acceptable salt thereof) in themanufacture of a medicament for inhibiting replication of HBV. Stillother embodiments described herein relate to one or more compounds ofFormula (I), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition that includes one or more compounds ofFormula (I), or a pharmaceutically acceptable salt form thereof, thatcan be used for inhibiting replication of HBV.

Embodiments of the present invention include a compound of Formula (I)as herein defined, or an enantiomer, diastereomer, solvate, or apharmaceutically acceptable salt form thereof, wherein the substituentsselected from one or more of the variables defined herein (e.g. R_(1A),R_(1B), R_(1C), B₁, R_(2A), R_(2B)) are independently selected to be anyindividual substituent or any subset of substituents from thoseexemplified in the listing in Table 1, below.

TABLE 1 Formula (I)

Cpd No R_(1A) R_(1B) R_(1C) B₁ R_(2A) R_(2B)  1 OCH₃ OH H b2 OH OH  2 OHOH H b2 OCH₃ OH  3 OCH₃ OH H b2 OCH₃ OH  4 F (*R)-SH H b2 OH (*R)-SH  5F (*S)-SH H b2 OH (*S)-SH  6 —O— OH CH₂ to form a b2 OH OH ring withR_(1A)  7 OH (*R)-BH₃ ⁻ H b2 OH (*R)-BH₃ ⁻  8 OH (*S)-BH₃ ⁻ H b2 OH(*S)-BH₃ ⁻  9 F (*R)-BH₃ ⁻ H b2 OCH₃ (*R)-SH 10 F (*S)-BH₃ ⁻ H b2 OCH₃(*R)-SH 11 F (*R)-BH₃ ⁻ H b2 OCH₃ (*R)-BH₃ ⁻ 12 F (*S)-BH₃ ⁻ H b2 OCH₃(*S)-BH₃ ⁻ 13 F (*R)-SH H b2 OCH₃ (*R)-BH₃ ⁻ 14 F (*S)-SH H b2 OCH₃(*S)-BH₃ ⁻ 15 F (*R)-BH₃ H b2 OCH₃ OH 16 F (*S)-BH₃ H b2 OCH₃ OH 17 F OHH b2 OCH₃ (*R)-BH₃ 18 F OH H b2 OCH₃ (*S)-BH₃ 19 —O— OH CH₂ to form a b2OCH₃ (*R)-BH₃ ring with R_(1A) 20 —O— (*R)-SH CH₂ to form a b2 OCH₃(*R)-BH₃ ring with R_(1A) 21 —O— (*R)-BH₃ CH₂ to form a b2 OCH₃ (*R)-BH₃ring with R_(1A) 22 —O— (*R)-BH₃ CH₂ to form a b2 OCH₃ OH ring withR_(1A) 23 —O— (*R)-BH₃ CH₂ to form a b2 OCH₃ (*R)-SH ring with R_(1A)

An embodiment of the present invention is directed to a compound ofFormula (I)

wherein

R_(1A) is hydroxy or fluoro and R_(1C) is hydrogen; or, R_(1A) is —O—and R_(1C) is CH₂ such that R_(1A) and R_(1C) are taken together withthe atoms to which they are attached to form a 5-membered ring;

R_(1B) is selected from the group consisting of hydroxy, thiol, and BH₃⁻;

B₁ is b2

R_(2A) is selected from the group consisting of hydroxy and methoxy;

R_(2B) is selected from the group consisting of hydroxy, thiol, and BH₃⁻;

provided that the compound of Formula (I) is other than(1R,6R,8R,9R,10R,15R,17R,18R)-17-(2-Amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-8-(6-amino-9H-purin-9-yl)-9-fluoro-3,12,18-trihydroxy-2,4,7,11,13,16-hexaoxa-3λ⁵,12λ⁵-diphosphatricyclo[13.2.1.0⁶,¹⁰]octadecane-3,12-dione, bis-ammoniumsalt; or an enantiomer, diastereomer, or pharmaceutically acceptablesalt form thereof.

A further embodiment of the present invention is directed to a compoundof Formula (I), selected from compounds 1 to 23,

or a pharmaceutically acceptable salt form thereof.

For use in medicine, salts of compounds of Formula (I) refer tonon-toxic “pharmaceutically acceptable salts.” Other salts may, however,be useful in the preparation of compounds of Formula (I) or of theirpharmaceutically acceptable salt forms thereof. Suitablepharmaceutically acceptable salts of compounds of Formula (I) includeacid addition salts that can, for example, be formed by mixing asolution of the compound with a solution of a pharmaceuticallyacceptable acid such as, hydrochloric acid, sulfuric acid, fumaric acid,maleic acid, succinic acid, acetic acid, benzoic acid, citric acid,tartaric acid, carbonic acid or phosphoric acid. Furthermore, where thecompounds of Formula (I) carry an acidic moiety, suitablepharmaceutically acceptable salts thereof may include alkali metal saltssuch as, sodium or potassium salts; alkaline earth metal salts such as,calcium or magnesium salts; and salts formed with suitable organicligands such as, quaternary ammonium salts. Thus, representativepharmaceutically acceptable salts include acetate, benzenesulfonate,benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calciumedetate, camsylate, carbonate, chloride, clavulanate, citrate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate,hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide,isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate,mesylate, methylbromide, methylnitrate, methylsulfate, mucate,napsylate, nitrate, N-methylglucamine ammonium salt, oleate, pamoate(embonate), palmitate, pantothenate, phosphate/diphosphate,polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate,tannate, tartrate, teoclate, tosylate, triethiodide, and valerate.

Representative acids and bases that may be used in the preparation ofpharmaceutically acceptable salts include acids including acetic acid,2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginicacid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoicacid, 4-acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid,(+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylicacid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid,2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaricacid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucoronicacid, L-glutamic acid, a-oxo-glutaric acid, glycolic acid, hippuricacid, hydrobromic acid, hydrochloric acid, (+)-L-lactic acid,(±)-DL-lactic acid, lactobionic acid, maleic acid, (−)-L-malic acid,malonic acid, (±)-DL-mandelic acid, methanesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid,orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid,L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebaicacid, stearic acid, succinic acid, sulfuric acid, tannic acid,(+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid andundecylenic acid; and bases including ammonia, L-arginine, benethamine,benzathine, calcium hydroxide, choline, deanol, diethanolamine,diethylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylenediamine,N-methyl-glucamine, hydrabamine, 1H-imidazole, L-lysine, magnesiumhydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassiumhydroxide, 1-(2-hydroxyethyl)-pyrrolidine, sodium hydroxide,triethanolamine, tromethamine, and zinc hydroxide.

Embodiments of the present invention include prodrugs of compounds ofFormula (I). In general, such prodrugs will be functional derivatives ofthe compounds that are readily convertible in vivo into the requiredcompound. Thus, in the methods of treating or preventing embodiments ofthe present invention, the term “administering” encompasses thetreatment or prevention of the various diseases, conditions, syndromesand disorders described with the compound specifically disclosed or witha compound that may not be specifically disclosed, but which converts tothe specified compound in vivo after administration to a patient.Conventional procedures for the selection and preparation of suitableprodrug derivatives are described, for example, in “Design of Prodrugs”,ed. H. Bundgaard, Elsevier, 1985.

Where the compounds according to embodiments of this invention have atleast one chiral center, they may accordingly exist as enantiomers.Where the compounds possess two or more chiral centers, they mayadditionally exist as diastereomers. It is to be understood that allsuch isomers and mixtures thereof are encompassed within the scope ofthe present invention. Furthermore, some of the crystalline forms forthe compounds may exist as polymorphs and as such are intended to beincluded in the present invention. In addition, some of the compoundsmay form solvates with water (i.e., hydrates) or common organicsolvents, and such solvates are also intended to be encompassed withinthe scope of this invention. The skilled artisan will understand thatthe term compound as used herein, is meant to include solvated compoundsof Formula (I).

Where the processes for the preparation of the compounds according tocertain embodiments of the invention give rise to mixture ofstereoisomers, these isomers may be separated by conventional techniquessuch as, preparative chromatography. The compounds may be prepared inracemic form, or individual enantiomers may be prepared either byenantiospecific synthesis or by resolution. The compounds may, forexample, be resolved into their component enantiomers by standardtechniques such as, the formation of diastereomeric pairs by saltformation with an optically active acid such as,(−)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-l-tartaric acidfollowed by fractional crystallization and regeneration of the freebase. The compounds may also be resolved by formation of diastereomericesters or amides, followed by chromatographic separation and removal ofthe chiral auxiliary. Alternatively, the compounds may be resolved usinga chiral HPLC column.

One embodiment of the present invention is directed to a composition,including a pharmaceutical composition, comprising, consisting of,and/or consisting essentially of the (+)-enantiomer of a compound ofFormula (I) wherein said composition is substantially free from the(−)-isomer of said compound. In the present context, substantially freemeans less than about 25%, preferably less than about 10%, morepreferably less than about 5%, even more preferably less than about 2%and even more preferably less than about 1% of the (−)-isomer calculatedas

${\%( + )\text{-}{enantiomer}} = {\frac{( {{{mass}( + )}\text{-}{enantiomer}} )}{( {{{mass}( + )}\text{-}{enantiomer}} ) + ( {{{mass}( - )}\text{-}{enantiomer}} )} \times 100.}$

Another embodiment of the present invention is a composition, includinga pharmaceutical composition, comprising, consisting of, and/orconsisting essentially of the (−)-enantiomer of a compound of Formula(I) wherein said composition is substantially free from the (+)-isomerof said compound. In the present context, substantially free from meansless than about 25%, preferably less than about 10%, more preferablyless than about 5%, even more preferably less than about 2% and evenmore preferably less than about 1% of the (+)-isomer calculated as

${\%( - )\text{-}{enantiomer}} = {\frac{( {{{mass}( - )}\text{-}{enantiomer}} )}{( {{{mass}( + )}\text{-}{enantiomer}} ) + ( {{{mass}( - )}\text{-}{enantiomer}} )} \times 100.}$

During any of the processes for preparation of the compounds of thevarious embodiments of the present invention, it may be necessary and/ordesirable to protect sensitive or reactive groups on any of themolecules concerned. This may be achieved by means of conventionalprotecting groups such as those described in Protective Groups inOrganic Chemistry, Second Edition, J. F. W. McOmie, Plenum Press, 1973;T. W. Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis,John Wiley & Sons, 1991; and T. W. Greene & P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, Third Edition, John Wiley & Sons, 1999. Theprotecting groups may be removed at a convenient subsequent stage usingmethods known from the art.

Even though the compounds of embodiments of the present invention(including their pharmaceutically acceptable salts and pharmaceuticallyacceptable solvates) can be administered alone, they will generally beadministered in admixture with a pharmaceutically acceptable carrier, apharmaceutically acceptable excipient and/or a pharmaceuticallyacceptable diluent selected with regard to the intended route ofadministration and standard pharmaceutical or veterinary practice. Thus,particular embodiments of the present invention are directed topharmaceutical and veterinary compositions comprising compounds ofFormula (I) and at least one pharmaceutically acceptable carrier,pharmaceutically acceptable excipient, and/or pharmaceuticallyacceptable diluent.

By way of example, in the pharmaceutical compositions of embodiments ofthe present invention, the compounds of Formula (I) may be admixed withany suitable binder(s), lubricant(s), suspending agent(s), coatingagent(s), solubilizing agent(s), and combinations thereof.

Solid oral dosage forms such as, tablets or capsules, containing thecompounds of the present invention may be administered in at least onedosage form at a time, as appropriate. It is also possible to administerthe compounds in sustained release formulations.

Additional oral forms in which the present inventive compounds may beadministered include elixirs, solutions, syrups, and suspensions; eachoptionally containing flavoring agents and coloring agents.

Alternatively, compounds of Formula (I) can be administered byinhalation (intratracheal or intranasal) or in the form of a suppositoryor pessary, or they may be applied topically in the form of a lotion,solution, cream, ointment or dusting powder. For example, they can beincorporated into a cream comprising, consisting of, and/or consistingessentially of an aqueous emulsion of polyethylene glycols or liquidparaffin. They can also be incorporated, at a concentration of betweenabout 1% and about 10% by weight of the cream, into an ointmentcomprising, consisting of, and/or consisting essentially of a wax orsoft paraffin base together with any stabilizers and preservatives asmay be required. An alternative means of administration includestransdermal administration by using a skin or transdermal patch.

The pharmaceutical compositions of the present invention (as well as thecompounds of the present invention alone) can also be injectedparenterally, for example, intracavernosally, intravenously,intramuscularly, subcutaneously, intradermally, or intrathecally. Inthis case, the compositions will also include at least one of a suitablecarrier, a suitable excipient, and a suitable diluent.

For parenteral administration, the pharmaceutical compositions of thepresent invention are best used in the form of a sterile aqueoussolution that may contain other substances, for example, enough saltsand monosaccharides to make the solution isotonic with blood.

In addition to the above described routes of administration for thetreatment of cancer, the pharmaceutical compositions may be adapted foradministration by intratumoral or peritumoral injection. The activationof the immune system in this manner to kill tumors at a remote site iscommonly known as the abscopal effect and has been demonstrated inanimals with multiple therapueutic modalities, (van der Jeught, et al.,Oncotarget, 2015, 6(3), 1359-1381). A further advantage of local orintratumoral or peritumoral administration is the ability to achieveequivalent efficacy at much lower doses, thus minimizing or eliminatingadverse events that may be observed at much higher doses (Marabelle, A.,et al., Clinical Cancer Research, 2014, 20(7), 1747-1756).

For buccal or sublingual administration, the pharmaceutical compositionsof the present invention may be administered in the form of tablets orlozenges, which can be formulated in a conventional manner.

By way of further example, pharmaceutical compositions containing atleast one of the compounds of Formula (I) as the active ingredient canbe prepared by mixing the compound(s) with a pharmaceutically acceptablecarrier, a pharmaceutically acceptable diluent, and/or apharmaceutically acceptable excipient according to conventionalpharmaceutical compounding techniques. The carrier, excipient, anddiluent may take a wide variety of forms depending upon the desiredroute of administration (e.g., oral, parenteral, etc.). Thus, for liquidoral preparations such as, suspensions, syrups, elixirs and solutions,suitable carriers, excipients and diluents include water, glycols, oils,alcohols, flavoring agents, preservatives, stabilizers, coloring agentsand the like; for solid oral preparations such as, powders, capsules,and tablets, suitable carriers, excipients and diluents includestarches, sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like. Solid oral preparations also may beoptionally coated with substances such as, sugars, or be entericallycoated so as to modulate the major site of absorption anddisintegration. For parenteral administration, the carrier, excipientand diluent will usually include sterile water, and other ingredientsmay be added to increase solubility and preservation of the composition.Injectable suspensions or solutions may also be prepared utilizingaqueous carriers along with appropriate additives such as, solubilizersand preservatives.

A therapeutically effective amount of a compound of Formula (I) or apharmaceutical composition thereof includes a dose range from about 0.01mg to about 3000 mg, or any particular amount or range therein, inparticular from about 0.05 mg to about 1000 mg, or any particular amountor range therein, or, more particularly, from about 0.05 mg to about 250mg, or any particular amount or range therein, of active ingredient in aregimen of about 1 to about 4 times per day for an average (70 kg)human; although, it is apparent to one skilled in the art that thetherapeutically effective amount for a compound of Formula (I) will varyas will the diseases, syndromes, conditions, and disorders beingtreated.

For oral administration, a pharmaceutical composition is preferablyprovided in the form of tablets containing about 1.0, about 10, about50, about 100, about 150, about 200, about 250, and about 500 milligramsof a compound of Formula (I).

Advantageously, a compound of Formula (I) may be administered in asingle daily dose, or the total daily dosage may be administered individed doses of two, three and four times daily.

Optimal dosages of a compound of Formula (I) to be administered may bereadily determined and will vary with the particular compound used, themode of administration, the strength of the preparation and theadvancement of the viral infection, disease, syndrome, condition ordisorder. In addition, factors associated with the particular subjectbeing treated, including subject gender, age, weight, diet and time ofadministration, will result in the need to adjust the dose to achieve anappropriate therapeutic level and desired therapeutic effect. The abovedosages are thus exemplary of the average case. There can be, of course,individual instances wherein higher or lower dosage ranges are merited,and such are within the scope of this invention.

Compounds of Formula (I) may be administered in any of the foregoingcompositions and dosage regimens or by means of those compositions anddosage regimens established in the art whenever use of a compound ofFormula (I) is required for a subject in need thereof.

As STING protein agonists, the compounds of Formula (I) are useful inmethods for treating or preventing a viral infection, disease, asyndrome, a condition or a disorder in a subject, including an animal, amammal and a human in which the viral infection, disease, the syndrome,the condition or the disorder is affected by the modulation, includingagonism, of the STING protein. Such methods comprise, consist of and/orconsist essentially of administering to a subject, including an animal,a mammal, and a human, in need of such treatment or prevention, atherapeutically effective amount of a compound, salt or solvate ofFormula (I).

In one embodiment, the present invention is directed to a compound ofFormula (I), or a pharmaceutically acceptable salt form thereof, for theuse in the treatment of cancer, and cancer diseases and conditions, or aviral infection.

Examples of cancer diseases and conditions for which compounds ofFormula (I), or pharmaceutically acceptable salts or solvates thereof,may have potentially beneficial antitumor effects include, but are notlimited to, cancers of the lung, bone, pancreas, skin, head, neck,uterus, ovaries, stomach, colon, breast, esophagus, small intestine,bowel, endocrine system, thyroid gland, parathyroid gland, adrenalgland, urethra, prostate, penis, testes, ureter, bladder, kidney orliver; rectal cancer; cancer of the anal region; carcinomas of thefallopian tubes, endometrium, cervix, vagina, vulva, renal pelvis, renalcell; sarcoma of soft tissue; myxoma; rhabdomyoma; fibroma; lipoma;teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemagioma;hepatoma; fibrosarcoma; chondrosarcoma; myeloma; chronic or acuteleukemia; lymphocytic lymphomas; primary CNS lymphoma; neoplasms of theCNS; spinal axis tumors; squamous cell carcinomas; synovial sarcoma;malignant pleural mesotheliomas; brain stem glioma; pituitary adenoma;bronchial adenoma; chondromatous hanlartoma; inesothelioma; Hodgkin'sDisease or a combination of one or more of the foregoing cancers.Suitably the present invention relates to a method for treating orlessening the severity of cancers selected from the group consisting ofbrain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme,Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease,Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma,medulloblastoma, head and neck, kidney, liver, melanoma, ovarian,pancreatic, adenocarcinoma, ductal madenocarcinoma, adenosquamouscarcinoma, acinar cell carcinoma, glucagonoma, insulinoma, prostate,sarcoma, osteosarcoma, giant cell tumor of bone, thyroid, lymphoblasticT cell leukemia, chronic myelogenous leukemia, chronic lymphocyticleukemia, hairy-cell leukemia, acute lymphoblastic leukemia, acutemyelogenous leukemia, chronic neutrophilic leukemia, acute lymphoblasticT cell leukemia, plasmacytoma, Immunoblastic large cell leukemia, mantlecell leukemia, multiple myeloma, megakaryoblastic leukemia, multiplemyeloma, acute megakaryocytic leukemia, pro myelocytic leukemia,erythroleukemia, malignant lymphoma, hodgkins lymphoma, non-hodgkinslymphoma, lymphoblastic T cell lymphoma, Burkitt's lymphoma, follicularlymphoma, neuroblastoma, bladder cancer, urothelial cancer, vulvalcancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma,esophageal cancer, salivary gland cancer, hepatocellular cancer, gastriccancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST(gastrointestinal stromal tumor) and testicular cancer.

In another embodiment, the present invention is directed to a compoundof Formula (I), or a pharmaceutically acceptable salt form thereof, foruse in the treatment of a disorder affected by the agonism of STINGselected from the group consisting of melanoma, colon cancer, breastcancer, prostate cancer, lung cancer, fibrosarcoma, and hepatitis B.

The disclosed compounds of Formula (I) may be useful in combination withone or more additional compounds useful for treating HBV infection.These additional compounds may comprise other disclosed compounds and/orcompounds known to treat, prevent, or reduce the symptoms or effects ofHBV infection. Such compounds include, but are not limited to, HBVpolymerase inhibitors, interferons, viral entry inhibitors, viralmaturation inhibitors, literature-described capsid assembly modulators,reverse transcriptase inhibitors, immunomodulatory agents, TLR-agonists,and other agents with distinct or unknown mechanisms that affect the HBVlife cycle or that affect the consequences of HBV infection.

In non-limiting examples, the disclosed compounds may be used incombination with one or more drugs (or a salt thereof) selected from thegroup comprising:

HBV reverse transcriptase inhibitors, and DNA and RNA polymeraseinhibitors including, but not limited to, lamivudine (3TC, Zeffix,Heptovir, Epivir, and Epivir-HBV), entecavir (Baraclude, Entavir),adefovir dipivoxil (Hepsara, Preveon, bis-POM PMEA), tenofovirdisoproxil fumarate (Viread, TDF or PMPA);

interferons including, but not limited to, interferon alpha (IFN-α),interferon beta (IFN-β), interferon lambda (IFN-λ), and interferon gamma(IFN-γ);

viral entry inhibitors;

viral maturation inhibitors;

capsid assembly modulators, such as, but not limited to, BAY 41-4109;

reverse transcriptase inhibitors;

immunomodulatory agents such as TLR-agonists; and

agents of distinct or unknown mechanisms, such as, but not limited to,AT-61((E)-N-(1-chloro-3-oxo-l-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)benzamide),AT-130((E)-N-(1-bromo-1-(2-methoxyphenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-2-yl)-4-nitrobenzamide),and analogs thereof.

In one embodiment, the additional therapeutic agent is an interferon.The term “interferon” or “IFN” refers to any member of the family ofhighly homologous species-specific proteins that inhibit viralreplication and cellular proliferation and modulate immune response.

For example, human interferons are grouped into three classes: Type I,which includes interferon-alpha (IFN-α), interferon-beta (IFN-β), andinterferon-omega (IFN-ω), Type II, which includes interferon-gamma(IFN-γ), and Type III, which includes interferon-lambda (IFN-λ).Recombinant forms of interferons that have been developed and arecommercially available are encompassed by the term “interferon” as usedherein. Subtypes of interferons, such as chemically modified or mutatedinterferons, are also encompassed by the term “interferon” as usedherein. Chemically modified interferons may include pegylatedinterferons and glycosylated interferons. Examples of interferons alsoinclude, but are not limited to, interferon-alpha-2a,interferon-alpha-2b, interferon-alpha-n1, interferon-beta-1a,interferon-beta-1b, interferon-lamda-1, interferon-lamda-2, andinterferon-lamda-3. Examples of pegylated interferons include pegylatedinterferon-alpha-2a and pegylated interferon alpha-2b.

Accordingly, in one embodiment, the compounds of Formula (I) can beadministered in combination with an interferon selected from the groupconsisting of interferon alpha (IFN-α), interferon beta (IFN-β),interferon lambda (IFN-λ), and interferon gamma (IFN-γ). In one specificembodiment, the interferon is interferon-alpha-2a, interferon-alpha-2b,or interferon-alpha-n1. In another specific embodiment, theinterferon-alpha-2a or interferon-alpha-2b is pegylated. In a preferredembodiment, the interferon-alpha-2a is pegylated interferon-alpha-2a(PEGASYS). In another embodiment, the additional therapeutic agent isselected from immune modulator or immune stimulator therapies, whichincludes biological agents belonging to the interferon class.

Further, the additional therapeutic agent may be an agent that disruptsthe function of other essential viral protein(s) or host proteinsrequired for HBV replication or persistence.

In another embodiment, the additional therapeutic agent is an antiviralagent that blocks viral entry or maturation or targets the HBVpolymerase such as nucleoside or nucleotide or non-nucleos(t)idepolymerase inhibitors. In a further embodiment of the combinationtherapy, the reverse transcriptase inhibitor or DNA or RNA polymeraseinhibitor is Zidovudine, Didanosine, Zalcitabine, ddA, Stavudine,Lamivudine, Abacavir, Emtricitabine, Entecavir, Apricitabine,Atevirapine, ribavirin, acyclovir, famciclovir, valacyclovir,ganciclovir, valganciclovir, Tenofovir, Adefovir, PMPA, cidofovir,Efavirenz, Nevirapine, Delavirdine, or Etravirine.

In an embodiment, the additional therapeutic agent is animmunomodulatory agent that induces a natural, limited immune responseleading to induction of immune responses against unrelated viruses. Inother words, the immunomodulatory agent can effect maturation of antigenpresenting cells, proliferation of T-cells and cytokine release (e.g.,IL-12, IL-18, IFN-alpha, -beta, and -gamma and TNF-alpha among others),

In a further embodiment, the additional therapeutic agent is a TLRmodulator or a TLR agonist, such as a TLR-7 agonist or TLR-9 agonist. Infurther embodiment of the combination therapy, the TLR-7 agonist isselected from the group consisting of SM360320(9-benzyl-8-hydroxy-2-(2-methoxy-ethoxy)adenine) and AZD 8848 (methyl[3-({[3-(6-amino-2-butoxy-8-oxo-7,8-dihydro-9H-purin-9-yl)propyl][3-(4-morpholinyl)propyl]amino}methyl)phenyl]acetate).

In any of the methods provided herein, the method may further compriseadministering to the individual at least one HBV vaccine, a nucleosideHBV inhibitor, an interferon or any combination thereof. In anembodiment, the HBV vaccine is at least one of RECOMBIVAX HB, ENGERIX-B,ELOVAC B, GENEVAC-B, or SHANVAC B.

In one embodiment, the methods described herein further compriseadministering at least one additional therapeutic agent selected fromthe group consisting of nucleotide/nucleoside analogs, entry inhibitors,fusion inhibitors, and any combination of these or other antiviralmechanisms.

In another aspect, provided herein is method of treating an HBVinfection in an individual in need thereof, comprising reducing the HBVviral load by administering to the individual a therapeuticallyeffective amount of a disclosed compound alone or in combination with areverse transcriptase inhibitor; and further administering to theindividual a therapeutically effective amount of HBV vaccine. Thereverse transcriptase inhibitor may be at least one of Zidovudine,Didanosine, Zalcitabine, ddA, Stavudine, Lamivudine, Abacavir,Emtricitabine, Entecavir, Apricitabine, Atevirapine, ribavirin,acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir,Tenofovir, Adefovir, PMPA, cidofovir, Efavirenz, Nevirapine,Delavirdine, or Etravirine.

In another aspect, provided herein is a method of treating an HBVinfection in an individual in need thereof, comprising reducing the HBVviral load by administering to the individual a therapeuticallyeffective amount of a disclosed compound alone or in combination with anantisense oligonucleotide or RNA interference agent that targets HBVnucleic acids; and further administering to the individual atherapeutically effective amount of HBV vaccine. The antisenseoligonucleotide or RNA interference agent possesses sufficientcomplementarity to the target HBV nucleic acids to inhibit replicationof the viral genome, transcription of viral RNAs, or translation ofviral proteins.

In another embodiment, the disclosed compound and the at least oneadditional therapeutic agent are co-formulated. In yet anotherembodiment, the disclosed compound and the at least one additionaltherapeutic agent are co-administered. For any combination therapydescribed herein, synergistic effect may be calculated, for example,using suitable methods such as the Sigmoid-E_(max) equation (Holford &Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22: 27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

In an embodiment of any of the methods of administering combinationtherapies provided herein, the method can further comprise monitoring ordetecting the HBV viral load of the subject, wherein the method iscarried out for a period of time including until such time that the HBVvirus is undetectable.

Abbreviations used in the instant specification, particularly theschemes and examples, are as follows:

ACN acetonitrile

AcOH glacial acetic acid

ADDP azodicarboxylic dipiperidide

aq. aqueous

Bn or Bzl benzyl

BINAP 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl

Boc tert-butyloxycarbonyl

conc. concentrated

dba dibenzylideneacetone

DBU 1,8-diazabicyclo[5.4.0]undec-7-ene

DCC N,N′-dicyclohexyl-carbodiimide

DCE 1,2-dichloroethane

DCM dichloromethane

DEAD diethyl azodicarboxylate

DIBAL diisobutylaluminum hydride

DIPEA or DIEA diisopropyl-ethyl amine

DMA dimethylaniline

DMAP 4-dimethylaminopyridine

DME dimethoxyethane

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

DMT 4,4′-dimethoxytrityl

DPPA diphenylphosphoryl azide

dppf 1,1′-bis(diphenylphosphino)ferrocene

EA ethyl acetate

EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

ESI electrospray ionization

EtOAc or EA ethyl acetate

EtOH ethanol

GCMS gas chromatography-mass spectrometry

h or hr(s) hour or hours

HEK human embryonic kidney

HPLC high performance liquid chromatography

LAH lithium aluminum hydride

LDA lithium diisopropylamide

LHMDS lithium bis(trimethylsilyl)amide

MEK methyl ethyl ketone

MeOH methanol

MHz megahertz

min minute or minutes

MS mass spectrometry

Ms methanesulfonyl

NBS N-bromosuccinimide

NIS N-iodosuccinimide

NMM N-methylmorpholine

NMP N-methylpyrrolidone

NMR nuclear magnetic resonance

PCC pyridinium chlorochromate

PE petrolum ether

RP reverse-phase

rt or RT room temperature

R_(t) retention time

Sec second or seconds

SEM-Cl 2-(trimethylsilyl)ethoxymethyl chloride

TBAF tetrabutylammonium fluoride

TBDMS t-butyldimethylsilyl

TBP tributyl phosphate

TEA or Et₃N triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

TIPS triisopropylsilyl

TLC thin layer chromatography

TMS tetramethylsilane

Ts 4-toluenesulfonyl

SPECIFIC EXAMPLES Example 1

Step 1: Preparation of Compound 1e

To a solution of 3′-O-methyl-guanosine 1d (CAS 10300-27-3, 1.0 g, 3.36mmol) in pyridine (20 mL) was added dropwisetert-butylchlorodimethylsilane (3.2 mL, 25.2 mmol) at room temperature.After 1 h, isobutyryl chloride (1.08 g, 10.1 mmol) was added dropwise atroom temperature. The final mixture was stirred at room temperature for2 h. The mixture was quenched with water (30 mL) at 0° C. and NH₄OH (6mL) was added dropwise at 0° C. After 10 min, the mixture was stirred atrt for 0.5 h. The mixture was concentrated. The crude product waspurified by FCC (DCM:MeOH=10:1) to afford 1e (790 mg, 63.9%) as a whitesolid. ¹H NMR (400 MHz, DMSO-d₆) 12.08 (s, 1H), 11.67 (s, 1H), 8.27 (s,1H), 5.81 (d, J=6.0 Hz, 1H), 5.51 (d, J=6.0 Hz, 1H), 5.10 (t, J=5.2 Hz,1H), 4.59-4.57 (m, 1H), 4.01-3.99 (m, 1H), 3.86-3.84 (m, 1H), 3.65-3.57(m, 2H), 3.41 (s, 3H), 3.17 (d, J=5.2 Hz, 1H), 2.79-2.76 (m, 1H), 1.14(s, 3H), 1.12 (s, 3H). ESI-MS: m/z=368.0[M+1]⁺.

Step 2: Preparation of Compound 1f

A solution of compound 1e (790 mg, 2.15 mmol) and DMTrCl (0.765 g, 2.26mmol) in pyridine (10 mL) was stirred at room temperature overnight.DMTCl (0.765 g, 2.26 mmol) was added and the reaction was stirred atroom temperature for 2 h. The mixture was quenched with water (10 mL)and extracted with DCM (10 mL×4). The combined organic layer was driedover Na₂SO₄, filtered and the filtrate concentrated. The residue waspurified by flash chromatography (DCM:MeOH=15:1, R_(F)=0.5) to affordcompound 1f (1.28 g, 88.9%) as a light yellow solid. ¹H NMR (400 MHz,CDCl₃) 11.87 (s, 1H), 7.68-7.66 (m, 2H), 7.57 (d, J=7.6 Hz, 2H), 7.44(t, J=9.2 Hz, 4H), 7.31-7.29 (m, 2H), 7.22-7.19 (m, 1H), 6.87-6.81 (m,4H), 5.70 (d, J=7.2 Hz, 1H), 5.30-5.27 (m, 1H), 5.05-5.03 (m, 1H),4.19-4.18 (m, 1H), 4.09-4.07 (m, 1H), 3.78 (s, 3H), 3.77 (s, 3H),3.58-3.55 (m, 1H), 3.47 (s, 3H), 3.05-3.03 (m, 1H), 1.47-1.40 (m, 1H),0.85 (d, J=6.8 Hz, 3H), 0.55 (d, J=6.8 Hz, 3H); ESI-MS: m/z=670.2[M+1]⁺.

Step 3: Preparation of Compound 1g

To a solution of compound 1f (1.28 g, 1.91 mmol) and DIPEA (741.0 mg,5.73 mmol) in THF (5 mL) was added3-((chloro(diisopropylamino)phosphino)oxy) propanenitrile (1.36 g, 5.73mmol) at room temperature. The mixture was stirred at room temperaturefor 1 h. The reaction was quenched with MeOH. The mixture was extractedwith EtOAc and the combined organic layers were washed with brine twice.The organic layer was dried over Na₂SO₄, filtered and the filtrateconcentrated. The residue was purified by flash column chromatography(DCM:MeOH=10:1, R_(f)=0.6) to afford compound 1g (1 g, 60.1%). ¹H NMR(400 MHz, CD₃CN) 7.88 (d, J=9.0 Hz, 1H), 7.48-7.41 (m, 2H), 7.35-7.24(m, 7H), 6.88-6.79 (m, 4H), 6.02-5.89 (m, 1H), 5.19-4.95 (m, 1H),4.28-4.20 (m, 1H), 4.07-4.04 (m, 1H), 3.78 (d, J=1.6 Hz, 7H), 3.67-3.48(m, 4H), 3.44 (d, J=15.6 Hz, 3H), 3.33 (td, J=2.8, 10.8 Hz, 1H),2.72-2.65 (m, 1H), 2.61-2.53 (m, 1H), 2.51 (t, J=6.0 Hz, 1H), 1.26-1.24(m, 4H), 1.18-1.12 (m, 12H), 0.91 (d, J=6.8 Hz, 3H); ³¹P NMR (162 MHz,CD3CN) 150.90 (s, 1P), 150.81 (s, 1P), 13.80 (s, 1P); ESI-MS: m/z787.2[M+1]⁺.

Step 4: Preparation of Compound 1b

To a solution of compound 1a (4.3 g, 4.51 mmol) and water (156.8 mg, 8.7mmol) in dry CH₃CN (16 mL) was added pyridinium trifluoroacetate (1.0 g,5.2 mmol) at room temperature. After 1 min, t-butylamine (4 mL) wasadded. The resulting mixture was stirred at 15° C. for 20 min. Themixture was concentrated for 2 h to afford the crude product 1b as awhite solid (4.0 g). The crude product was used directly for the nextstep.

Step 5: Preparation of Compound 1c

To a solution of compound 1b (4.05 g, 4.35 mmol) and water (832.0 mg,46.2 mmol) in DCM (40 mL) was added dichloroacetic acid (2.1 g, 16.3mmol) at room temperature for 50 min. After 10 min, pyridine (730.6 mg,9.24 mmol) was added. The mixture was concentrated and the residue waspurified by flash column chromatography (CH₂Cl₂:MeOH=5:1, R_(f)=0.4) toafford compound 1c (2.45 g, 89.5%) as a white solid.

ESI-MS: m/z=449.9 [M+1]⁺.

Step 6: Preparation of Compound 1i

A solution of compound 1c (300 mg, 0.48 mmol) and 4 Å molecular sievesin dry CH₃CN (20 mL) was stirred at room temperature under N₂ for 10min. 1H-imidazole perchlorate (1.5 g, 8.8 mmol) was added. After 10 min,compound 1g (0.54 g, 0.62 mmol) in dry CH₃CN (5 mL) was added. Themixture was stirred at room temperature for 50 min. tert-Butylhydroperoxide (0.43 mL, 2.39 mmol) was added. The resulting mixture wasstirred at room temperature for 1 h and concentrated. The mixture wasconcentrated and the residue was purified by preparative HPLC (water (10mM NH₄HCO₃)—CH₃CN) to afford compound 1i (168 mg, 34.1%) as a whitesolid. ¹H NMR (400 MHz, CD₃OD) 8.89 (s, 1H), 8.79 (s, 1H), 8.36 (s, 1H),8.16 (d, J=7.5 Hz, 2H), 7.73-7.67 (m, 1H), 7.62 (t, J=7.0 Hz, 2H),6.27-6.18 (m, 2H), 5.38-5.30 (m, 1H), 4.81 (m, 2H), 4.44 (s, 1H), 4.29(s, 1H), 4.26-4.15 (m, 3H), 3.92-3.85 (m, 1H), 3.75 (d, J=12.4 Hz, 1H),3.61-3.57 (m, 3H), 2.74 (td, J=6.6, 13.1 Hz, 1H), 1.21 (dd, J=6.8, 15.2Hz, 6H), 0.85 (s, 9H), 0.12 (s, 3H), −0.04 (s, 3H); ³¹P NMR (162 MHz,CD₃OD) δ3.61 (s, 1P), −1.68 (s, 1P); ESI-MS: m/z=1033.2 [M+1]⁺.

Step 7: Preparation of Compound 1k

To a solution of compound 1i (160 mg, 0.16 mmol) and 4 Å molecularsieves in pyridine (40 mL) was added DMOCP (87.0 mg, 0.47 mmol) at roomtemperature. The mixture was stirred at room temperature for 1 h. Iodine(199.4 mg, 0.79 mmol) and water (28.3 mg, 1.57 mmol) were added. After 1h, the mixture was filtered and then a saturated solution of Na₂SO₃ wasadded dropwise until the color of the filtrate changed to pale yellow.The mixture was filtered and the filtrate was concentrated. The residuewas purified by preparative HPLC (water (10 mM NH₄HCO₃)—CH₃CN from 23%to 53) to afford the target product 1k (48 mg, 31.3%) as a white solid.ESI-MS: m/z 977.5 [M+1]⁺.

Step 8: Preparation of Compound 1l

Compound 1k (40.0 mg, 0.041 mmol) was treated with a solution ofmethylamine in EtOH (33%, 10 mL) was stirred at room temperature for 1h. The reaction mixture was concentrated to give crude compound 1l (32.9mg, 100%) which was used directly for the next step. ESI-MS: m/z 803.4[M+1]⁺.

Step 9: Preparation of Compound 2

A solution of compound 1l (32.86 mg, 0.041 mmol), Et₃N (248.5 mg, 2.46mmol) and triethylamine trihydrofluoride(198.0 mg, 1.2 mmol) in pyridine(5 mL) was stirred at 50° C. for 5 h. The mixture was diluted with THF(10 mL) and isopropoxytriethylsilane (541.5 mg, 4.1 mmol) was added atroom temperature for 1.5 h. The mixture was concentrated and the residuewas purified by preparative HPLC (water (0.05% NH₄OH v/v)-CH₃CN from 0%to 15%) to afford the target product 2 as its ammonium salt (6.7 mg) asa white solid. ¹H NMR (400 MHz, D₂O) δ=8.18-8.16 (d, J=10 Hz, 2H), 7.74(s, 1H), 6.06 (s, 1H), 5.79-5.77 (d, J=8.8 Hz, 1H), 5.62-5.56 (m, 1H),4.99-4.94 (m, 1H), 4.45 (m, 1H), 4.39-4.34 (m, 2H), 4.15-4.03 (m, 4H),3.46 (s, 3H); ³¹P NMR (162 MHz, D₂O) −1.28 (s, 1P), −2.65 (s, 1P);ESI-MS: m/z 689.5 [M+1]⁺.

Step 9: Preparation of (Cpd 2, Na Salt)

A 3 mL volume of Dowex 50 W×8, 200-400 (H form) was added to a beaker(for 6.7 mg of compound 5 ammonium salt) and washed with deionized water(2×). Then to the resin was added 15% H₂SO₄ in deionized water (50 mL),the mixture was stirred for 15 min, and decanted (1×). The resin wastransferred to a column with 15% H₂SO₄ in deionized water and washedwith 15% H₂SO₄ (at least 4 CV), and then with deionized water until itwas neutral. The resin was transferred back into the beaker, and 15%NaOH in water solution (50 mL) was added, and the mixture was stirredfor 15 min, and decanted (1×). The resin was transferred to the columnand washed with 15% NaOH in water (at least 4 CV), and then with wateruntil it was neutral (at least 4 CV). Compound 5 (6.7 mg) was dissolvedin deionized water (6.7 mg in 1 mL) and added to the top of the column,and eluted with deionized water. The compound was eluted out in earlyfractions as detected by TLC (UV). The product was lyophilized to givetarget compound 2 Na salt (6.4 mg, 94.2%) as a white foam. ¹H NMR (400MHz, D₂O) 8.16 (s, 1H), 8.13 (s, 1H), 7.74 (s, 1H), 6.04 (s, 1H), 5.78(d, J=8.8 Hz, 1H), 5.61-5.56 (m, 1H), 4.98-4.93 (m, 1H), 4.65 (s, 1H),4.44-4.35 (m, 3H), 4.15-4.05 (m, 4H), 3.46 (s, 3H); ³¹P NMR (162 MHz,D₂O) −1.26, −2.64; ESI-MS: m/z 689.0 [M+1]⁺.

Example 2

Step 1: Preparation of Compound 2b

To a solution of DMT-2′-OMe-Bz-adenosine-CE phosphoramidite 2a (1.0 g,1.13 mmol) and water (40.6 mg, 2.25 mmol) in dry CH₃CN (4 mL) was addedpyridinium trifluoroacetate (261.0 mg, 1.35 mmol) at 15° C. To thereaction mixture was added t-butylamine (4 mL). The mixture wasconcentrated to afford 1 g of the crude compound 2b as a white solid,which was co-evaporated with DCM (3×) and used directly for the nextstep. ESI-MS: m/z=450.0 [M+1]⁺. (DMT=4,4′-dimethoxytrityl).

Step 2: Preparation of Compound 2c

To a solution of compound 2b (930.0 mg, 1.12 mmol) and water (0.2 g,11.2 mmol) in CH₂Cl₂ (10 mL) was added dichloroacetic acid (0.51 g, 4.0mmol) at 15° C. for 0.5 h. After 10 min, pyridine was added. The mixturewas concentrated and the residue was purified by flash columnchromatography (CH₂Cl₂:MeOH=5:1, R_(f)=0.5) to afford compound 2c (400mg, 0.76 mmol, 67.6% yield) as a white solid. ³¹P NMR (400 MHz, DMSO-d₆)δ0.05; ESI-MS: m/z=450.0 (M+1).

Step 3: Preparation of Compound 2f

A solution of compound 2c (860.0 mg, 1.63 mmol) and 4 Å molecular sieves(1 g) in dry CH₃CN (16 mL) was stirred at 15° C. under N₂ for 10 min.1H-Imidazole perchlorate (5.16 g, 30.26 mmol) was added. After 10 min,DMT-3′-O-TBDMS-G(iBu)-CE phosphoramidite 2d (2.05 g, 8.11 mmol) in dryCH₃CN (4 mL) was added. The mixture was stirred at room temperature for50 min. A solution of tert-butyl hydroperoxide (TBHP, 1.48 mL, 8.14mmol, 5.5 M in hexane) was added. The resulting mixture was stirred at15° C. for 1 h. The mixture was concentrated and the residue waspurified by preparative HPLC (water (10 mM NH₄HCO₃)—CH₃CN) to affordcompound 2f (600 mg, 0.58 mmol, 35.7% yield) as a white solid. ¹H NMR(400 MHz, CD₃OD) δ8.61 (d, J=5.2 Hz, 1H), 8.41-8.30 (m, 1H), 8.24-8.17(m, 1H), 8.04-7.90 (m, 2H), 7.61-7.49 (m, 2H), 7.48-7.40 (m, 2H),6.11-6.03 (m, 1H), 6.02-5.98 (m, 1H), 5.36-5.12 (m, 1H), 4.55-4.43 (m,2H), 4.41-4.32 (m, 1H), 4.30-4.19 (m, 2H), 4.13-4.02 (m, 1H), 3.99-3.85(m, 2H), 3.75-3.65 (m, 1H), 3.63-3.53 (m, 1H), 3.37 (s, 3H), 2.70-2.69(m, 1H), 2.60-2.52 (m, 2H), 1.10-1.03 (m, 6H), 0.82-0.77 (m, 9H),0.04-0.00 (m, 6H); ³¹P NMR (162 MHz, CD₃OD) δ3.17, 3.13, −2.58, −2.69;ESI-MS: m/z=517.1 [M/2+1]⁺ and 1032.3 [M+1]⁺.

Step 4: Preparation of Compound 2i+Compound 2j

To a solution of compound 2g (280.0 mg, 0.27 mmol) and 4 Å molecularsieves (1 g) in pyridine (60 mL) was added5,5-dimethyl-2-oxo-2-chloro-1,3,2-dioxa-phosphinane (DMOCP, 150.2 mg,0.81 mmol) at 16° C. The mixture was stirred at 16° C. for 1 h. Iodine(344.3 mg, 1.36 mmol) and water (48.9 mg, 2.71 mmol) were added. After 1h, the reaction was quenched with a saturated solution of Na₂SO₃. Themixture was filtered and the filtrate was concentrated. The residue waspurified by preparative HPLC (water (10 mM NH₄HCO₃)—CH₃CN) to afford amixture of compound 2i and compound 2j (170 mg, 0.17 mmol, 60.8% yield)as a white solid. ESI-MS: m/z=1030.4 [M+H]³⁰.

Step 5: Preparation of Compound 2j

A mixture of compound 2i and compound 2j (170 mg, 0.17 mmol) was treatedwith a solution of methylamine in EtOH (15 mL, 33%) and the resultingsolution was stirred at 15° C. for 1 h. The crude product 2j (134.3 mg)was used directly for the next step.

Step 6: Preparation of Compound 1

A solution of compound 2j (134.3 mg, crude), Et₃N (1.0 g, 10.0 mmol) andtriethylamine trihydrofluoride (Et₃N-3HF, 807.4 mg, 5.00 mmol) inpyridine (10 mL) was stirred at 50° C. for 5 h. The mixture was dilutedwith THF (10 mL) and isopropoxy trimethylsilane (2.2 g, 16.7 mmol) wasadded. After stirring at 15° C. for 1 h, the mixture was concentrated at15° C. and the residue was purified by preparative HPLC (water (0.05%NH₄OH v/v)—CH₃CN) to afford compound 1 as its ammonium salt (19.5 mg,0.028 mmol) as a white solid upon lyophilization.

¹H NMR (400 MHz, D₂O) δ8.26 (s, 1H), 8.15 (s, 1H), 7.78 (s, 1H), 6.116(s, 1H), 5.87 (d, J=8.4 Hz, 1H), 5.66 (s, 1H), 4.95 (s, 1H), 4.48 (d,J=4.4 Hz, 1H), 4.24 (m, 5H), 3.97 (d, J=11.7 Hz, 1H), 3.83 (d, J=12.0Hz, 1H), 3.69 (s, 3H); ³¹P NMR (162 MHz, D₂O) δ−1.57, −3.38; ESI-MS:m/z=688.9 [M+H]⁺.

Preparation of Compound 1, Sodium Salt

Dowex 50 W×8, 200-400 (25 mL, H form) was added to a beaker and washedwith deionized water (60 mL). Then to the resin was added 15% H₂SO₄ indeionized water, and the mixture was gently stirred for 5 min, anddecanted (50 mL). The resin was transferred to a column with 15% H₂SO₄in deionized water and washed with 15% H₂SO₄ (at least 4 CV), and thenwith deionized water until it was neutral. The resin was transferredback into the beaker, 15% NaOH in deionized water solution was added,and mixture was gently stirred for 5 min, and decanted (1×). The resinwas transferred to the column and washed with 15% NaOH in H₂O (at least4 CV), and then with deionized water until it was neutral. Compound 1,ammonium salt (16 mg) was dissolved in a minimum amount of deionizedwater, added to the top of the column, and eluted with deionized water.Appropriate fractions of CDN based on UV were pooled together andlyophilized to afford the sodium salt form of compound 1 (13.5 mg). ¹HNMR (400 MHz, D₂O) δ8.17 (s, 1H), 8.14 (s, 1H), 7.73 (s, 1H), 6.14 (s,1H), 5.83 (d, J=8.8 Hz, 1H), 5.58-5.52 (m, 1H), 5.01 (s, 1H), 4.98-4.90(m, 1H), 4.04-4.51 (m, 5H), 4.04 (d, J=11.7 Hz, 1H), 3.78 (d, J=12.0 Hz,1H), 3.69 (s, 3H); 31P NMR (162 MHz, D₂O) δ−1.63, −2.29; ESI-MS: m/z=689[M+H]+.

Example 3

Step 1: Preparation of Compound 3a

To a solution DMT-3′-O-TBDMS-G(iBu)-CE phosphoramidite compound 2d (1 g,1.03 mmol) and water (37.1 mg, 2.06 mmol) in CH₃CN (4 mL) was addedpyridinium trifluoroacetate (238.9 mg, 1.2 mmol) at room temperature. Tothe reaction mixture was added tert-butylamine (4 mL). The resultingmixture was stirred at room temperature for 20 min. The mixture wasconcentrated to afford compound 3a (941.1 mg) as a white solid, whichwas co-evaporated with DCM (3×) and used directly for the next step.

Step 2: Preparation of Compound 3b

To a solution of compound 3a (941.1 mg, 1.03 mmol) and water (0.19 g,10.0 mmol) in CH₂Cl₂ (30 mL) was added a solution of dichloroacetic acid(0.47 g, 3.62 mmol, 6% in DCM) at room temperature for 0.5 h. Pyridine(0.163 g, 2.06 mmol) was added. After 10 min, the mixture wasconcentrated and the residue was purified by flash column chromatography(DCM:MeOH=5:1, R_(f)=0.5) to afford 3c (515 mg, 0.84 mmol) as a whitesolid. ESI-MS m/z 532.1 [M+1]⁺.

Step 3: Preparation of Compound 3ea+Compound 3eb

A solution of compound 3b (500 mg, 0.82 mmol) and 4 Å MS (0.5 g) in dryCH₃CN (10 mL) was stirred at room temperature under N₂ for 3 min.1H-Imidazole perchlorate (IMP, 2.54 g, 15.1 mmol) was added. After 10min, LNA-dA(Bz)-CE phosphoramidite, compound 3c (943 mg, 1.06 mmol) inCH₃CN (5 mL) was added. The mixture was stirred at room temperature for50 min. A solution of tert-butyl hydroperoxide (TBHP, 5.5 M in hexane,0.74 mL, 4.09 mmol) was added. The resulting mixture was stirred at roomtemperature for 1 h. The mixture was concentrated and the residue waspurified by preparative HPLC (water (10 mM NH₄HCO₃)-ACN) to afford amixture of compound 3ea and compound 3eb (135.7 mg, 0.132 mmol) as awhite solid. The product mixture was used directly for the next step.ESI-MS m/z 1030.1 [M+1]⁺.

Step 4: Preparation of Compound 3g

To a solution of compound 3ea and compound 3eb (135.7 mg, 0.132 mmol)and 4 Å molecular sieves (0.5 g) in pyridine (30 mL) was added DMOCP(72.6 mg, 0.39 mmol) at 29° C. The mixture was stirred at 29° C. for 1h. Iodine (166.3 mg, 0.66 mmol) and water (23.6 mg, 1.31 mmol) wereadded. After 1 h, the reaction was quenched with a saturated solution ofNa₂SO₃. The mixture was filtered and the filtrate was concentrated. Theresidue was purified by preparative HPLC (water (10 mM NH₄HCO₃)—CH₃CNfrom 35% to 65%) to afford compound 3g (22 mg, 0.021 mmol) as a whitesolid. ESI-MS m/z 514.5 [M/2+1]⁺ and 1029.3 [M+1]⁺.

Step 5: Preparation of Compound 3h

Compound 3g (22 mg, 0.021 mmol) was treated with a solution ofmethylamine in EtOH (33%, 10 mL) was stirred at room temperature for 1h. The reaction mixture was concentrated to give crude compound 3h,which was co-evaporated with pyridine (3×) and used directly into thenext step.

Step 6: Preparation of Compound 6 Ammonium Salt

A solution of compound 3h, Et₃N (176.7 mg, 1.75 mmol) and triethylaminetrihydrofluoride (Et₃N-3HF, 140.7 mg, 0.87 mmol) in pyridine (10 mL) wasstirred at 50° C. for 5 h. The mixture was diluted with THF (10 mL) andisopropoxytrimethylsilane (384.9 mg, 2.91 mmol) was added at 15° C. for1 h. The mixture was concentrated at room temperature and the residuewas purified by preparative HPLC (water (0.05% NH₄OH v/v)—CH₃CN from 0%to 15%) to afford compound 6 as its ammonium salt (6.1 mg, 0.009 mmol)as a white solid upon lyophilization. ¹H NMR (400 MHz, D₂O) 8.30 (s,1H), 8.11 (s, 1H), 7.83 (brs, 1H), 6.19 (s, 1H), 5.96 (d, J=6.8 Hz, 1H),4.98 (s, 1H), 4.57 (s, 1H), 4.36-4.30 (m, 3H), 4.16 (d, J=6.0 Hz, 3H),4.04 (d, J=7.6 Hz, 1H), 3.86 (d, J=11.6 Hz, 2H); ³¹P NMR (162 MHz, D₂O)−1.73, −3.40; ESI-MS m/z 687.0 [M+1]⁻.

Preparation of Compound 6 Sodium Salt

Dowex 50W×8, 200-400 (2 mL, H form) was added to a beaker and washedwith deionized water (15 mL). Then to the resin was added 15% H₂SO₄ indeionized water, the mixture was gently stirred for 5 min, and decanted(10 mL). The resin was transferred to a column with 15% H₂SO₄ indeionized water and washed with 15% H₂SO₄ (at least 4 CV), and then withdeionized water until it was neutral. The resin was transferred backinto the beaker, 15% NaOH in deionized water solution was added, andmixture was gently stirred for 5 min, and decanted (1×). The resin wastransferred to the column and washed with 15% NaOH in water (at least 4CV), and then with deionized water until it was neutral. Compound 6,ammonium salt (3.5 mg) was dissolved in a minimum amount of deionizedwater, added to the top of the column, and eluted with deionized water.Appropriate fractions of CDN based on UV were pooled together andlyophilized to afford the sodium salt of compound 6 (3.02 mg). ¹H NMR(400 MHz, D₂O): δ8.11 (s, 1H), 7.88 (s, 1H), 7.74 (s, 1H), 6.02 (s, 1H),5.85 (d, J=8.4 Hz, 1H), 5.65-5.58 (s, 1H), 4.91 (d, J=3.2 Hz, 1H), 4.83(s, 1H), 4.50 (d, J=4.8 Hz, 1H), 4.35-4.28 (m, 1H), 4.26-4.19 (m, 2H),4.13-4.01 (m, 3H), 3.90 (d, J=8 Hz, 1H), 3.76 (d, J=12.4 Hz, 2H);³¹P-NMR (162 MHz, D₂O) δ−1.64, −1.91.

Example 4

Step 1: Preparation of Compound 4ba and Compound 4bb

A solution of compound 1c (300 mg, 0.57 mmol) and 4 Å molecular sieves(0.5 g) in dry CH₃CN (10 mL) was stirred at 29° C. under N₂ for 3 min.1H-Imidazole perchlorate (1.76 g, 10.5 mmol) was added. After 10 min, asolution of compound 1g (500 mg, 0.58 mmol) in dry CH₃CN (10 mL) wasadded. The mixture was stirred at room temperature for 50 min andtert-butyl hydroperoxide (TBHP, 0.52 mL, 2.84 mmol) was added. Theresulting mixture was stirred at 29° C. for 1 h. The mixture wasconcentrated and the residue was purified by preparative HPLC (water (10mM NH₄HCO₃)—CH₃CN) to afford a mixture of compounds 4ba and 4bb (100 mg,0.114 mmol) as a white solid. ³¹P NMR (162 MHz, DMSO) −0.66, −2.44;ESI-MS: m/z 932.3 (M+1).

Step 2: Preparation of Compound 4da and Compound4db

To a suspension of compound 4ba and compound 4bb (100.0 mg, 0.11 mmol)and 4 Å molecular sieves (0.5 g) in pyridine (30 mL) was added DMOCP(59.4 mg, 0.32 mmol) at 28° C. The mixture was stirred at 28° C. for 1h. Iodine (136.2 mg, 0.54 mmol) and water (19.3 mg, 1.1 mmol) wereadded. After 1 h, the reaction was quenched with a saturated solution ofNa₂SO₃. The mixture was filtered and the filtrate was concentrated. Theresidue was purified by preparative HPLC (water (10 mM NH₄HCO₃)-ACN from1% to 28%) to afford a mixture of compounds 4da and 4db (50 mg, 0.057mmol) as a white solid. The product was used directly for the next step.

Step 3: Preparation of Compound 3, Ammonium Salt

A mixture of compounds 4da and 4db (50 mg, 0.057 mmol) was treated witha solution of methylamine in EtOH (33%, 20 mL) and the mixture wasstirred at room temperature for 2 h. The reaction mixture wasconcentrated to give crude compound 3, which was purified by preparativeHPLC (water (0.05% NH₄OH v/v)—CH₃CN from 0% to 10%) to give compound 3ammonium salt as a white solid (16 mg, 0.023 mmol). ³¹P NMR (162 MHz,D₂O) −1.53, −3.41.

The product was further purified by preparative HPLC (water (0.05% NH₄OHv/v)—CH₃CN from 0% to 10%) to afford compound 3 as its ammonium salt, asa white solid.

Step 4: Preparation of Compound 3, Sodium Salt

Compound 3 ammonium salt was dried under high vacuum to give a whitesolid (12 mg). Dowex 50W×8, 200-400 (H form, 3 mL) was added to a beaker(for 12 mg of compound 6) and washed with deionized water (2×). Then tothe resin was added 15% H₂SO₄ in deionized water (50 mL) and the mixturewas stirred for 15 min and decanted (1×). The resin was transferred to acolumn with 15% H₂SO₄ in deionized water and washed with 15% H₂SO₄ (atleast 4 CV), and then with deionized water until it was neutral. Theresin was transferred back into the beaker, and 15% NaOH in deionizedwater solution (50 mL) was added and the mixture was stirred for 15 minand decanted (1×). The resin was transferred to the column and washedwith 15% NaOH in deionized water (at least 4 CV), and then withdeionized water until it was neutral (at least 4 CV). Compound 3 wasdissolved in deionized water (12 mg in 1 mL), added to the top of thecolumn, and eluted with deionized water. The converted sodium salt waseluted out in early fractions as detected by TLC (UV). The product waslyophilized to give compound 3, sodium salt (7.4 mg, 0.010 mmol). ¹H NMR(400 MHz, D₂O) δppm 8.17 (s, 1H), 8.14 (s, 1H), 7.74 (s, 1H), 6.14 (s,1H), 5.79 (d, J=8.8 Hz, 1H), 5.65-5.59 (m, 1H), 5.02-5.00 (m, 1H), 4.44(s, 1H), 4.36-4.29 (m, 3H), 4.15-4.11 (m, 3H), 4.04-4.01 (m, 1H), 3.66(s, 3H), 3.46 (s, 3H); ³¹P NMR (162 MHz, D₂O) −1.53, −2.62; ESI-MS m/z702.5 (M+1).

Example 5

Step 1: Preparation of Compound 5b

To a solution of DMT-2′-F-dA(Bz)-CE phosphoramidite 5a (2.20 g, 2.51mmol) in CH₃CN (12.0 mL) was added water (90.5 mg, 5.02 mmol, 2.0 eq)and pyridinium trifluoroacetate (582.1 mg, 3.01 mmol, 1.2 eq). Themixture was stirred at 25° C. for 5 min. Then tert-butylamine (12.0 mL)was added and the reaction mixture was stirred at 25° C. for 15 min. Themixture was concentrated under reduced pressure to give a foam, whichwas dissolved in CH₃CN (10.0 mL) and concentrated again to affordcompound 5b (1.69 g, 2.29 mmol, 91.0% yield) as a white foam. ESI-MS:m/z 740.2 [M+H]⁺.

Step 2: Preparation of Compound 5c

To a solution of compound 5b (1.69 g, 2.29 mmol) in CH₂Cl₂ (24.0 mL) wasadded water (411.8 mg, 21.9 mmol, 10.0 eq) and a solution of 2,2-dichloroacetic acid (6% in DCM, 24 mL) slowly. The mixture was stirredat 25° C. for 0.5 h. The reaction was quenched with pyridine (2 mL) andthe reaction mixture was concentrated to afford a residue, which waspurified by column chromatography on silica gel (DCM/MeOH=10/1 to 5/1)to afford compound 5c (856 mg, 1.62 mmol, 70.9% yield) as a white foam.

Step 3: Preparation of Compound 5e

To a solution of compound 5c (380 mg, 0.70 mmol) in CH₃CN (12.0 mL) wasadded 4 Å molecular sieves (0.5 g), the resulting mixture was stirred at25° C. for 10 min. 1H-Imidazole perchlorate (IMP, 356.7 mg, 2.1 mmol,3.0 eq) was added and the mixture stirred for an additional 10 minbefore DMT-3′-O-TBDMS-G(iBu)-CE phosphoramidite 2d ((J. Am. Chem. Soc.2001,123, 8165-8176), 811.6 mg, 0.84 mmol, 1.2 eq) was added. Themixture was stirred at 25° C. for 1 h to afford a solution of compound5d (a solution in CH₃CN), thenN,N-dimethyl-N′-(5-sulfanylidene-1,2,4-dithiazol-3-yl)methanimidamide(DDTT, 715.7 mg, 3.49 mmol, 5 eq.) was added to above reaction mixtureat 25° C. and stirred for 1 h at the same temperature. The reactionmixture was filtered, and the filtrate was concentrated under reducedpressure to afford a residue, which was purified by preparative HPLC(H₂O—CH₃CN) to afford compound 5e (130.0 mg, 0.125 mmol, 18.0% yieldover two steps) as a white solid. ESI-MS: m/z 1036.1 [M+H]⁺.

Step 4: Preparation of Compound 5f

To a solution of compound 5e (130.0 mg, 0.125 mmol) in pyridine (24 mL)was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (DMOCP,69.5 mg, 0.38 mmol, 3.0 eq) at 25° C., the mixture was stirred at 25° C.for 1 h to afford a solution of compound 5f (127.7 mg, 0.125 mmol, 100%yield, a solution in pyridine) which was used for the next step withoutfurther purification.

Step 5: Preparation of Compound 5ga+Compound 5gb

To a solution of compound 5f (127.7 mg, 0.125 mmol) in pyridine wasadded 3H-benzo[c][1,2]dithiol-3-one (211.1 mg, 1.26 mmol, 10 eq) at 25°C., and the resulting mixture was stirred at 25° C. for 1 h. Thereaction mixture was filtered, and the filtrate was concentrated underreduced pressure to afford a residue, which was purified by preparativeHPLC (water (0.225% formic acid)-CH₃CN) to afford compound 5ga (22.0 mg,0.021 mmol, 18.1% yield over two steps) as a white solid and compound5gb (55.0 mg, 0.052 mmol, 45.2% yield over two steps) as a white solid.ESI-MS: m/z 1050.2 [M+H]⁺, 525.8 [M/2+H]⁺ (compound 5ga). ESI-MS: m/z1050.2 [M+H]⁺, 525.6 [M/2+H]⁺(compound 5gb).

Step 6: Preparation of Compound 5hb

Compound 5gb (55.0 mg, 0.052 mmol, 1.00 eq) was treated with a solutionof methylamine (3.00 mL, 35% in EtOH) and the resulting mixture wasstirred at 25° C. for 12 h. The reaction mixture was concentrated underreduced pressure to afford compound 5hb (39.0 mg, 0.047 mmol, 90.5%yield) which was used for the next step without further purification.ESI-MS: m/z 823.1 [M+H]⁺.

Step 7: Preparation of Compound 4, Ammonium Salt

To a solution of compound 5hb (44.0 mg, 0.053 mmol) in pyridine (13.0mL) was added Et₃N (324.7 mg, 3.2 mmol, 60 eq) and triethylaminetrihydrofluoride (258.6 mg, 1.6 mmol, 30 eq) at 25° C., and the mixturewas stirred at 50° C. for 5 h, then isopropoxytrimethylsilane (707.4 mg,5.3 mmol, 100 eq) was added at 15° C. and stirred for 1 h. The mixturewas concentrated at 15° C. and the residue was purified by preparativeHPLC (water (0.05% NH₄OH v/v)—CH₃CN) to afford compound 4 as itsammonium salt (6.0 mg, 0.008 mmol, 15.8% yield) as a white solid. ¹NMR(400 MHz, D₂O) δ8.33 (s, 1H), 8.25 (s, 1H), 7.85 (s, 1H), 6.45 (d,J=14.3 Hz, 1H), 5.92 (d, J=8.5 Hz, 1H), 5.77 (s, 1H), 5.51 (s, 0.5H),5.38 (s, 0.5H), 5.23 (d, J=21.5 Hz, 1H), 4.53-4.42 (m, 5H), 4.10 (d,J=11.3 Hz, 1H), 3.99 (d, J=12.8 Hz, 1H); ¹⁹F NMR (376 MHz, D₂O)□−122.94(br, s, 1F); ³¹P NMR (162 MHz, D₂O) □ 55.99 (brs, 1P), 51.19 (brs, 1P);ESI-MS: m/z 708.9[M+H]⁺.

Step 6a: Preparation of Compound 5ha

Compound 5ga (13.0 mg, 0.012 mmol, 1.00 eq) was treated with a solutionof methylamine (1.00 mL, 35% in EtOH) and the solution was stirred at25° C. for 12 h. The reaction mixture was concentrated under reducedpressure to afford compound 5ha (9.0 mg, 0.011 mmol, 88.4% yield) whichwas used for the next step without further purification. ESI-MS: m/z823.3 [M+H]⁺.

Step 7a: Preparation of Compound 5, Ammonium Salt

To a solution of compound 5ha (39.0 mg, 0.047 mmol) in pyridine (7.0 mL)was added Et₃N (287.8 mg, 2.84 mmol, 60 eq) and triethylaminetrihydrofluoride (229.2 mg, 1.42 mmol, 30 eq) at 25° C., the mixture wasstirred at 50° C. for 5 h, then isopropoxytrimethylsilane (627.0 mg,4.74 mmol, 100 eq) was added and at 15° C. and stirred for 1 h. Themixture was concentrated at 15° C. and the residue was purified bypreparative HPLC (water (0.05% NH₄OH v/v)—CH₃CN) to afford compound 5 asits ammonium salt (6.60 mg, 0.009 mmol, 19.6% yield) as a white solid.¹H NMR (400 MHz, D₂O) δ8.54 (s, 1H), 8.25 (s, 1H), 7.84 (s, 1H), 6.46(d, J=13.8 Hz, 1H), 5.94 (d, J=8.3 Hz, 1H), 5.81-5.75 (m, 1H), 5.54 (d,J=2.8 Hz, 0.5H), 5.41 (d, J=3.0 Hz, 0.5H), 5.30-5.23 (m, 1H), 4.54-4.40(m, 5H), 4.09-4.04 (m, 2H); ¹⁹F NMR (376 MHz, D₂O) δ−201.92 (brs, 1F);³¹P NMR (162 MHz, D₂O) δ55.97 (s, 1P), 53.90 (brs, 1P); MS: m/z 708.9[M+H]⁺.

Step 8: Preparation of Compound 4, Sodium Salt

Dowex 50W×8, 200-400 (3 mL, H form) was added to a beaker and washedwith deionized water (15 mL). Then to the resin was added 15% H₂SO₄ indeionized water, the mixture was gently stirred for 5 min, and decanted(10 mL). The resin was transferred to a column with 15% H₂SO₄ indeionized water and washed with 15% H₂SO₄ (at least 4 CV), and then withdeionized water until it was neutral. The resin was transferred backinto the beaker, 15% NaOH in deionized water solution was added, andmixture was gently stirred for 5 min, and decanted (1×). The resin wastransferred to the column and washed with 15% NaOH in water (at least 4CV), and then with deionized water until it was neutral. Compound 4,ammonium salt (6.9 mg) was dissolved in a minimal amount of deionizedwater, added to the top of the column, and eluted with deionized water.Appropriate fractions of CDN based on UV were pooled together andlyophilized to afford the sodium salt form of compound 4 (6.45 mg). ¹HNMR (400 MHz, D₂O) δ8.41 (s, 1H), 8.12 (s, 1H), 7.73 (s, 1H), 6.35 (d,J=13.6 Hz, 1H), 5.83 (d, J=8.4 Hz, 1H), 5.72-5.68 (m, 1H), 5.43 (d,J=2.8 Hz, 0.5 H), 5.30 (d, J=2.8 Hz, 0.5 H), 5.18-5.10 (m, 1H),4.65-4.29 (m, 5H), 4.05-3.93 (m, 2H); ¹⁹F NMR (376 MHz, D₂O) δ−201.76;³¹P NMR (162 MHz, D₂O) □ 55.88, 53.91.

Step 9: Preparation of Compound 5, Sodium Salt

Dowex 50W×8, 200-400 (3 mL, H form) was added to a beaker and washedwith deionized water (15 mL). Then to the resin was added 15% H₂SO₄ indeionized water, the mixture was gently stirred for 5 min, and decanted(10 mL). The resin was transferred to a column with 15% H₂SO₄ indeionized water and washed with 15% H₂SO₄ (at least 4 CV), and then withdeionized water until it was neutral. The resin was transferred backinto the beaker, 15% NaOH in deionized water solution was added, andmixture was gently stirred for 5 min, and decanted (1×). The resin wastransferred to the column and washed with 15% NaOH in water (at least 4CV), and then with deionized water until it was neutral. Compound 5,ammonium salt (6.0 mg) was dissolved in minimal amount of deionizedwater, added to the top of the column, and eluted with deionized water.Appropriate fractions of CDN based on UV were pooled together andlyophilized to afford the sodium salt form of compound 5 (5.12 mg). ¹HNMR (400 MHz, D₂O) □ 8.15 (s, 1H), 8.12 (s, 1H), 7.72 (s, 1H), 6.34 (d,J=14.4 Hz, 1H), 5.81 (d, J=8 Hz, 1H), 5.65-5.58 (m, 1H), 5.45 (d, J=3.2Hz, 0.5H), 5.32 (d, J=3.6 Hz, 0.5H), 5.20-5.05 (m, 1H), 4.65-4.29 (m,5H), 3.90-4.04 (m, 2H); ¹⁹F NMR (376 MHz, D₂O) δ−201.92; ³¹P NMR (162MHz, D₂O) □□055.74, 53.23; MS: m/z 709.00 [M+H]+.

Example 6

Step 1: Preparation of Compound 6d

The nucleoside compound 6a (1.63 g, 2.12 mmol) was co-evaporated with amixture of anhydrous toluene: anhydrous acetonitrile (1:1, v/v, 3×20mL), then dissolved in anhydrous acetonitrile (50 mL) andphosphoramidite 6b (2.1 gr, 2.12 mmol). 4 Å molecular sieves powder (4.0gr) were added to this. The resulting heterogeneous mixture was bubbledwith Argon gas for 4 min. After stirring this mixture at rt for 30 min,0.45 M tetrazole in acetonitrile (30 mL, 12.72 mmol) was added at rt.After stirring the reaction for 45 min, reaction mixture was filteredthen washed with sat. aq. NaHCO₃ (1×20 mL) and sat. aq. NaCl (1×20 mL),dried over MgSO₄, and the filtrate evaporated to dryness to affordphosphite compound 6c, which was used directly without furtherpurification for the next step.

The crude phosphite compound 6c was dissolved in anhydrous CH₂Cl₂ (40mL), then 4 Å molecular sieves powder (4.0 gr) was added to this. Theresulting heterogeneous mixture was bubbled with Argon gas for 4 min.After stirring this mixture at rt for 30 min, borane dimethyl sulfidecomplex solution (2.0 M in THF, BH₃-DMS, 3.49 mL, 6.99 mmol) was addedvery slowly over 5 min at 0° C. After stirring the reaction for 20 minat rt, the reaction mixture quickly filtered, then diluted EtOAc (120mL), quenched with water (20 mL). The phases were separated and theorganic phase was washed with water (1×20 mL), sat. aq. NaCl (1×20 mL),and then the aqueous phase was back-extracted with EtOAc (1×20 mL). Thecombined organic phases were evaporated to dryness, and the resultingcrude material was purified by flash column chromatography on silica gel(0-85% EtOAc in hexane, v/v) to afford boranophosphate dimer 6d (980mg). ESI-MS: m/z 1670.30 [M+H]⁺.

Step 2: Preparation of Compound 6e

Di-DMTr-boranophosphate dimer 6d (2.3 gr, 1.37 mmol) was dissolved in80% aq. AcOH:CH₃CN (3:1, v/v, 13 mL). After stirring the reactionmixture for 16 h at 37° C., the mixture was diluted with EtOAc (70 mL),then washed sequentially with sat. aq. NaHCO₃ (3×20 mL) and sat. aq.NaCl (1×15 mL). The organic phase was evaporated to dryness, resultingin a crude residue which was purified by flash column chromatographyover silica gel (20-100% Acetone in Hexane, v/v) to afforddiol-boranophosphate dimer 6e (0.9 g). ESI-MS: m/z 1066.45 [M+H]⁺.

Step 3: Preparation of Compound 6f

The diol nucleoside compound 6e (0.55 g, 0.516 mmol) was co-evaporatedwith a mixture of anhydrous toluene: anhydrous acetonitrile (1:1, v/v,3×20 mL) then dissolved in anhydrous acetonitrile (20 mL) and 4 Åmolecular sieves powder (1.0 g) was added to this. The resultingheterogeneous mixture was bubbled with Argon gas for 4 min. Afterstirring this mixture at rt for 30 min, 0.45 M tetrazole in acetonitrile(7 mL, 3.09 mmol) was added at rt, then after stirring the reaction for75 min, the mixture was filtered, the filtrate washed sequentially withsat. aq. NaHCO₃ (1×20 mL) and sat. aq. NaCl (1×20 mL), dried over MgSO₄(stirred for 5 min then filtered) and evaporated to dryness to affordcompound 6f. The resulting mixture was used directly without furtherpurification for the next step. The crude phosphite 6f was dissolved inanhydrous CH₂Cl₂ (20 mL), then 4 Å molecular sieves powder (1.0 g) wasadded to this. The resulting heterogeneous mixture was bubbled withArgon gas for 4 min. After stirring this mixture at rt for 30 min,borane dimethyl sulfide complex solution (2.0 M in THF, BH₃-DMS, 0.93mL, 1.84 mmol) was added very slowly over 5 min at 0 □C. After stirringthe reaction at rt, the reaction mixture quickly filtered, then dilutedwith EtOAc (80 mL), and quenched with water (20 mL). The phases werepartitioned and the organic phase was washed with sat. aq. NaCl (1×20mL), then the aqueous phase was back-extracted with EtOAc (1×20 mL). Thecombined organic phases were evaporated to dryness, the resulting crudematerial was purified by flash column chromatography on silica gel(0-10% MeOH in dichloromethane, v/v) to afford fully-protected cyclicboranophosphate 6f (480 mg, 75% pure). ESI-MS: m/z 1179.73 [M+H]⁺.

Step 4: Preparation of Compound 6g

3′-Silyl-G(iBu)-2′-silyl-A(Bz)-2′,3′-cyclicdinucleotide-boranophosphate6f (437 mg, approximately 75% pure) was dissolved in mixture of aq.ammonia: EtOH (7 mL, 3:1, v/v). After stirring the reaction mixture for16 h at 50 □C, the reaction mixture was concentrated to dryness andco-evaporated with EtOH (2×10 mL) and toluene (2×20 mL). The resultingcrude solid was washed with dichloromethane (40 mL) and the precipitatewas collected by filtration to afford the di-TBS protected cyclic dimer6g (ESI-MS: m/z 896.20 [M−H]⁻), which was used for the next reactionwithout any further purification.

To remove the TBS groups, cyclic dimer compound 6g (350 mg crude) wasdissolved in anhydrous DMSO (5.5 mL) and to this it was addedtriethylamine trihydrofluoride (HF.3TEA, 2.8 mL and trimethylamine 0.6mL). After stirring the reaction mixture for 3.5 h at 50° C., it wasneutralized with triethylamine and purified by preparative HPLC (BufferA: 50 mM triethylammonium acetate in H₂O; Buffer B: 50 mMtriethylammoniumacetate in CH₃CN, gradient: 0-30% of B over 30 min, flowrate 24 mL/min) to afford two isomers of boranophosphate 6i (9 mg) and6j (4 mg) as a triethylammonium salt as a white solid. ESI-MS: m/z 668.6[M−J]⁻.

Step 5: Preparation of Compound 7 and Compound 8 as Sodium Salt.

Dowex 50W×8, 200-400 (3 mL, H form) was added to a beaker and washedwith deionized water (20 mL). Then to the resin was added 15% H₂SO₄ indeionized water, the mixture was gently stirred for 5 min, and decanted(10 mL). The resin was transferred to a column with 15% H₂SO₄ indeionized water and washed with 15% H₂SO₄ (at least 4 CV), and then withdeionized water until it was neutral. The resin was transferred backinto the beaker, 15% NaOH in deionized water solution was added, andmixture was gently stirred for 5 min, and decanted (1×). The resin wastransferred to the column and washed with 15% NaOH in water (at least 4CV), and then with deionized water until it was neutral. Thetriethylammonium form of both isomers of cyclic boranophosphates 6i (9mg) and 6j (4 mg) was dissolved in minimum amount of deionized water,added to the top of the column, and eluted with deionized water.Appropriate fractions of CDN based on UV were pooled together andlyophilized to afford the sodium salt form of compound 7 (8.2 mg) andcompound 8 (3.3 mg), respectively.

Compound 7: ¹H NMR (400 MHz, D₂O): □ 8.15 (s, 1H), 8.09 (s, 1H), 7.99(s, 1H), 6.01 (s, 1H), 5.93 (d, J=8.7 Hz, 1H), 4.98-4.93 (m, 1H),4.87-4.80 (m, 1H), 4.74-4.60 (m, 1H), 4.42-4.37 (m, 2H), 4.31 (s, 1H),4.24-4.15 (m, 2H), 3.92-3.82 (m, 2H), 0.5 to −0.5 (very broad peak, 6H);³¹P NMR (162 MHz, D₂O) 94.70 (very broad peak); ESI-MS: m/z 668.6[M−1]⁻.

Compound 8: ¹H NMR (400 MHz, D₂O): δ8.12 (s, 1H), 8.10 (s, 1H), 7.95 (s,1H), 5.99 (d, J=2.4 Hz, 1H), 5.92 (d, J=8.4 Hz, 1H), 5.22-5.15 (m, 1H),4.83-4.76 (m, 1H), 4.75-4.71 (m, 1H), 4.50 (d, J=4 Hz, 1H), 4.40-4.30(m, 1H), 4.31 (s, 1H), 4.25-4.18 (m, 1H), 4.15-4.10 (m, 1H), 4.02-3.96(m, 1H), 3.90-3.84 (m, 1H), −0.2 to 0.9 (very broad peak, 6H); ³¹P NMR(162 MHz, D₂O) δ93.75 (very broad peak); ESI-MS: m/z: 668.7 [M−1]⁻.

Example 7

Step 1: Preparation of Compound 7c

The nucleoside compound 1f (2.3 g, 3.43 mmol) was co-evaporated with amixture of anhydrous toluene/anhydrous acetonitrile (1:1, v/v, 3×50 mL),then dissolved in anhydrous acetonitrile (80 mL). 4 Å Molecular sievespowder (4.0 g) and tetrazole in acetonitrile (61 mL, 0.45 M, 27.44 mmol)were added to the reaction mixture. The resulting heterogeneous mixturewas purged with bubbling Ar_((g)) for 4 min. After stirring at rt for 10min, a solution of amidite 7a (3.0 g, 3.43 mmol, ChemGenes Corp.) inanhydrous acetonitrile (15 mL) was added at rt. After stirring for 1 h45 min at rt, the reaction mixture was diluted with ethyl acetate (250mL), filtered, and the filtrate washed with saturated aqueous NaHCO₃(1×40 mL) and brine (1×40 mL). The filtrate was then dried (MgSO₄),stirred for 5 min, filtered, and the filtrate concentrated to dryness toafford phosphite 7b (ESI-MS: m/z 1444.45 [M+1]⁺).

Crude phosphite compound 7b was used in the next step without furtherpurification. Crude compound 7b was dissolved in anhydrous pyridine (100mL) and(E)-N,N-dimethyl-N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide(DDTT, 2.12 g, 10.29 mmol) was added at rt. After stirring for 1 h atrt, the reaction mixture was diluted with ethyl acetate (250 mL), washedsequentially with saturated aqueous NaHCO₃ (1×40 mL) and brine (1×40mL), and concentrated to dryness. The aqueous phase was extracted withethyl acetate (1×20 mL). The combined organic phases were concentratedto dryness under reduced pressure to afford a crude material which waspurified by flash chromatography on silica gel (0-8% MeOH in CH₂Cl₂,v/v) to afford di-DMTr-phosphorothioate dimer 7c (4.6 g, ˜92%,approximately 90% purity). ESI-MS: m/z 1476.90 [M+H]⁺.

Step 2: Preparation of Compound 7d

Dimer 7c (4.5 g, 3.05 mmol) was dissolved in 80% aqueousAcOH:acetonitrile (90 mL, 8:2, v/v). After stirring the reaction mixturefor 20 h at 45° C., the mixture was diluted with ethyl acetate (400 mL)and washed sequentially with saturated aqueous NaHCO₃ (3×80 mL) andbrine (1×50 mL). The aqueous phase was separated and extracted withethyl acetate (1×20 mL). The combined organic phases were concentratedto dryness under reduced pressure and the resulting crude residue waspurified by flash chromatography on silica gel (0-15% MeOH in CH₂Cl₂,v/v) to afford dimer 7d (1.65 g, 63%). ESI-MS: m/z 872.10 [M+H]⁺.

Step 3: Preparation of Compound 7f

Dimer compound 7d (275 mg, 0.315 mmol) was co-evaporated with a mixtureof anhydrous toluene/anhydrous acetonitrile (1:1, v/v, 3×10 mL) thendissolved in anhydrous acetonitrile (20 mL, sonicated for 5 min forcomplete solubility of cpd 7d). 4 Å Molecular sieves powder (0.6 g) andtetrazole in acetonitrile (5.6 mL, 0.45 M, 2.52 mmol) were then added.The resulting heterogeneous mixture was purged with bubbling Ar_((g))for 4 min. After stirring the mixture at rt for 10 min, 2-cyanoethylN,N-diisopropylchlorophosphoramidite (142 mg, 0.473 mmol, 1.5 eq) wasadded at rt in five portions over 20 min. After stirring for 90 min atrt, the reaction mixture was diluted with ethyl acetate (60 mL),filtered, and the filtrate washed sequentially with saturated aqueousNaHCO₃ (1×20 mL) and brine (1×20 mL). The filtrate was then dried(MgSO₄) while stirring for 5 min, filtered, and the filtrateconcentrated to dryness under reduced pressure to afford compound 7e(ESI-MS: m/z 971.10 [M+1]⁺). The resulting residue was used in the nextstep without further purification.

Crude compound 7e was dissolved in anhydrous dichloromethane (25 mL), towhich was added 4 Å molecular sieves powder (0.5 g). The resultingheterogeneous mixture was purged with bubbling Ar_((g)) for 4 min. Uponstirring the mixture at rt for 10 min, the mixture was cooled to 0° C. Aborane dimethyl sulfide complex solution (2.0 M in THF, BH₃-DMS, 550 μL,3.5 eq) was added very slowly over 5 min at 0° C., and the reactionmixture was stirred at rt for 12 min. The mixture was then quicklyfiltered, diluted with ethyl acetate (80 mL), and quenched with water(20 mL). The organic phase was washed with brine (1×20 mL), and theaqueous layer was extracted with ethyl acetate (1×20 mL). The combinedorganic layers were concentrated to dryness under reduced pressure toafford a crude residue, which was purified by flash chromatography onsilica gel (0-10% MeOH in dichloromethane, v/v) to afford a mixture ofdiastereoisomers 7f (130 mg, ˜42% for 2 steps). ESI-MS: m/z 984.95[M+H]⁺.

Step 4: Preparation of Compound 7g and Compound 7h

The mixture of diastereoisomers 7f (130 mg) was dissolved in a mixtureof aqueous ammonia/ethanol (7 mL, 3:1, v/v). After stirring the reactionmixture for 18 h at 50° C., the reaction mixture was concentrated todryness and co-evaporated with ethanol (2×10 mL) and toluene (2×20 mL).The resulting crude solid was washed with dichloromethane (15 mL),collected by filtration, and purified by reverse phase preparative HPLC(column: Synergi 4μ, Hydro RP, 250 mm×30 mm, Mobile Phase:Buffer A:50 mMtriethylammonium acetate in H₂O; Buffer B: 50 mM triethylammoniumacetatein CH₃CN, gradient: 0-30% of B over 30 min, flow rate 24 mL/min) toafford a first minor boranophosphothioate isomer 7g (8.7 mg) and asecond major boranophosphothioate isomer 7h (13.1 mg) astriethylammonium acetate (TEAA) salts. ESI-MS: m/z: 703.1 [M−1]⁻.

Step 5: Preparation of Compound 9 and Compound 10

Dowex 50W×8, 200-400 (5 mL, H form) was added to a beaker and washedwith deionized water (30 mL). Then to the resin was added 15% H₂SO₄ indeionized water, the mixture was gently stirred for 5 min, then decanted(30 mL). The resin was transferred to a column with 15% H₂SO₄ indeionized water and washed with 15% H₂SO₄ (at least 4 CV), and then withdeionized water until neutral pH 7 was attained. The resin wastransferred back into the beaker, 15% NaOH in deionized water solutionwas added, the mixture was gently stirred for 5 min, then decanted (1×).The resin was transferred to the column and washed with 15% NaOH inwater (at least 4 CV), then with deionized water until neutral pH 7 wasattained. Each boranophosphothioate isomer 7g (8.7 mg) and 7h (13.1 mg)TEAA salts was dissolved in a minimum amount of deionized water, addedto the top of the column, and eluted with deionized water. Appropriatefractions of compounds 9 and 10 were pooled together and lyophilized toafford respectively compound 10 (7.8 mg) and compound 9 (12.4 mg) as asodium salt.

Compound 9 (major isomer): ¹H NMR (400 MHz, D₂O): δ8.07 (s, 1H), 7.97(s, 1H), 7.86 (s, 1H), 6.25 (d, J=16.4 Hz, 1H), 5.86 (d, J=8.4 Hz, 1H),5.52 (d, J=3.6 Hz, 0.5H), 5.40 (d, J=3.6 Hz, 0.5H) 5.16-5.19 (m, 1H),4.83-4.89 (m, 1H), 4.41-4.45 (m, 2H), 4.25-4.32 (m, 2H), 4.06-4.17 (m,2H), 3.88-3.94 (m, 1H), 3.46 (s, 3H), −0.1 to 0.65 (very broad peak,3H); ³¹P NMR (162 MHz, D₂O): δ94-95 (very broad peak, boranophosphate),52.59 (phosphorothiate); ¹⁹F NMR (379 MHz, D₂O): δ−201.7 (multiplet);ESI-MS: m/z: 703.1 [M−1]⁻.

Compound 10 (minor isomer): ¹H NMR (400 MHz, D₂O): δ8.18 (s, 1H), 8.13(s, 1H), 8.07 (s, 1H), 6.31 (d, J=15.6 Hz, 1H), 5.91 (d, J=8.4 Hz, 1H),5.65 (d, J=2.8 Hz, 0.5H), 5.50 (d, J=2.8 Hz, 0.5H) 5.07-5.30 (m, 2H),4.40-4.48 (m, 2H), 4.20-4.35 (m, 2H), 4.10-4.14 (m, 1H), 3.96-4.00 (m,1H), 3.83-3.88 (m, 1H), 3.48 (s, 3H), 0.2 to 0.8 (very broad peak, 3H);³¹P NMR (162 MHz, D₂O): δ94-95 (very broad peak, boranophosphate), 57.79(phosphorothiate); ¹⁹F NMR (379 MHz, D₂O): δ−202.4 (multiplet); ESI-MS:m/z: 703.1 [M−1]⁻.

Example 8

Step 1: Preparation of Compound 8a

To a solution of compound 7b (8 g, 5.538 mmol) in CH₂Cl₂ (80 mL) wasadded 4 Å molecular sieves powder (8 g) and the resulting heterogeneousmixture was bubbled with argon for 4 min. After stirring at rt for 30min, borane dimethyl sulfide complex solution (2.0 M in THF, BH₃-DMS,9.138 mL, 18.277 mmol) was added very slowly over 5 min at 0° C. Afterstirring the reaction for 2 h at rt, the reaction mixture was quicklyfiltered, diluted with CH₂Cl₂ (40 mL) and quenched with water (50 mL).The phases were separated and the organic layer was successively washedwith water (1×50 mL), brine (1×50 mL) and the aqueous layersback-extracted with CH₂Cl₂ (1×50 mL). The combined organic layers wereconcentrated under reduced pressure to dryness and the resulting crudematerial was purified by flash column chromatography on silica gel(petroleum ether/EtOAc=1/0˜0/1) to give compound 8a (7.5 g, 5.143 mmol)as a pale yellow oil.

Step 2: Preparation of Compound 8b

Compound 8a (7.5 g, 5.143 mmol) was added to a solution of CH₃CN (20 mL)in 80% acetic acid aqueous solution (60 mL) (CH₃CN/acetic acid aqueoussolution=1/3, acetic acid 48 mL, H₂O 12 mL, CH₃CN 20 mL). After stirringthe mixture reaction overnight at 25° C., triethylsilane was added andthe reaction stirred at 25° C. for an additional 1 h. The reactionmixture was quickly filtered, diluted with EtOAc (50 mL) and quenchedwith water (20 mL). The pH was adjusted to 7-8 with aqueous saturatedNaHCO₃ solution. The phases were separated and the organic layer wassuccessively washed with water (1×100 mL), brine (1×100 mL) andconcentrated under reduced pressure to dryness. The residue was purifiedby flash column chromatography on silica gel (petroleum/EtOAc=0/1 thenCH₂Cl₂/MeOH=1/0 to 20/1) to give compound 8b (6.5 g, 7.126 mmol) as awhite solid. ESI-MS: m/z=854.1 [M+1]⁺; ¹H NMR (400 MHz, DMSO-d₆) δ12.07(br d, J=13.5 Hz, 1H), 11.56 (d, J=9.7 Hz, 1H), 11.22 (br d, J=7.1 Hz,1H), 8.72 (d, J=3.1 Hz, 1H), 8.56-8.41 (m, 1H), 8.27-8.18 (m, 1H), 8.02(br d, J=6.2 Hz, 2H), 7.65-7.60 (m, 1H), 7.56-7.50 (m, 2H), 6.35 (br t,J=19.0 Hz, 1H), 6.00 (d, J=6.4 Hz, 1H), 5.95 (t, J=6.5 Hz, 1H),5.65-5.43 (m, 1H), 5.34-5.20 (m, 2H), 4.75-4.55 (m, 1H), 4.16-3.97 (m,6H), 3.92-3.82 (m, 1H), 3.67-3.49 (m, 2H), 3.14 (d, J=5.1 Hz, 3H),2.81-2.66 (m, 3H), 1.08 (br d, J=6.8 Hz, 6H), 0.59-−0.18 (m, 3H); ³¹PNMR (162 MHz, DMSO-d₆) 115.80 (br s, 1P).

Step 3: Preparation of Compound 8c

Compound 8b (1g, 1.096 mmol) was co-evaporated with CH₃CN (3×20 mL) anddissolved in anhydrous CH₃CN (44 mL). It was then added to 4 Å molecularsieves powder (1000 mg) and after stirring the mixture for 30 min, asolution of tetrazole in CH₃CN (0.45 M, 14.6 mL, 6 eq) was added at rt.The resulting mixture was stirred for 15 min at rt. It was then addeddropwise a solution of3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (495.647 mg,1.644 mmol) in anhydrous CH₃CN (5.0 mL) over 20 min. After stirring thereaction mixture for 1 h, an additional solution of tetrazole in CH₃CN(0.45 M, 9.7 mL, 4 eq) was added to this mixture at rt. The reactionmixture was filtered and the filtrate extracted with EtOAc (3×400 mL).The combined organic layers were successively washed with aqueous NaHCO₃(3×200 mL), brine (3×200 mL), dried over anhydrous Na₂SO₄, filtered, andthe filtrate concentrated under reduced pressure. The residue waspurified by flash column chromatography on silica gel (CH₂Cl₂: MeOH 100:0 to CH₂Cl₂:MeOH 90:10) to afford compound 8c (600 mg, 0.63 mmol) as awhite solid.

Step 4: Preparation of Compound 8d

To a solution of compound 8c (600 mg 0.63 mmol) in CH₂Cl₂ (15 mL) wasadded a solution of 2 M borane-dimethyl sulfide complex in THF (1039.276μL, 2.079 mmol) very slowly over 5 min at 0° C. After stirring thereaction mixture at 0° C. for 15 min, the reaction was quenched withMeOH (20 mL) at 0° C., then concentrated under reduced pressure. Thereaction was repeated a second time using the same scale. The two crudebatches were combined and purified by flash column chromatography onsilica gel (gradient eluent: CH₂Cl₂:MeOH 100:0 to CH₂Cl₂:MeOH 90: 10) togive compound 8d (1 g, 1.035 mmol, combined batches) as a white solid.ESI-MS: m/z=966.4 [M+1]⁺.

Step 5: Preparation of Compounds 11 and 12

Compound 8d (1.0 g, 1.035 mmol) was treated with a solution of MeNH₂ inEtOH (33%, 10 mL). After stirring at room temperature for 3 h, thereaction mixture was concentrated under reduced pressure and the residuewas purified by preparative high-performance liquid chromatography (PrepHPLC condition:Column:Phenomenex Kinetex XB-C18 150 mm×30 mm, 5 μm;Conditions: H₂O (A)-CH₃CN (B); Begin B: 0; End B: 20; Flow Rate: 25mL/min). The pure fractions were collected and lyophilized to dryness togive crude compound 11 (0.11 g, 0.160 mmol) and crude compound 12 (0.14g, 0.204 mmol) as white solids.

The crude compounds 11 and 12 were further purified by preparativehigh-performance liquid chromatography (Prep HPLCconditions:Column:DuraShell 150×25 mm×5 um; Condition: water (10 mMNH₄HCO₃) (A)-CH₃CN (B); Begin B: 0; End B: 15; Flow Rate: 35 mL/min).The pure fractions were collected and lyophilized to dryness to affordcompound 11 (0.08 g, 0.117 mmol) and compound 12 (0.04 g, 0.058 mmol),each as a white solid.

Compound 11: ¹H NMR (400 MHz, D₂O) δ8.21 (d, J=13.2 Hz, 2H), 8.05 (s,1H), 6.36 (d, J=16.3 Hz, 1H), 5.93 (d, J=8.2 Hz, 1H), 5.73-5.55 (m, 1H),5.19-5.02 (m, 2H), 4.55-4.48 (m, 2H), 4.34-4.24 (m, 2H), 4.16 (d, J=4.4Hz, 1H), 4.05-3.94 (m, 2H), 3.57 (s, 3H), 0.76-0.13 (m, 3H), -0.32 (brs, 3H); ESI-MS: m/z=686.9 [M+1]⁺; ¹⁹F NMR (376 MHz, D₂O)−202.02 (td,J=20.0, 50.3 Hz, 1F); ³¹P NMR (162 MHz, D₂O) δppm-94.42 (br s, 1P).

Compound 12: ¹H NMR (400 MHz, D₂O) δ8.31 (s, 1H), 8.23 (s, 1H), 7.86 (brs, 1H), 6.40 (br d, J=15.4 Hz, 1H), 5.89 (br d, J=8.6 Hz, 1H), 5.64-5.47(m, 1H), 5.35 (br s, 1H), 5.00-4.86 (m, 1H), 4.54-4.45 (m, 2H), 4.38 (brd, J=11.7 Hz, 1H), 4.20 (br d, J=17.0 Hz, 2H), 4.07 (br d, J=8.2 Hz,1H), 3.96 (br d, J=10.1 Hz, 1H), 3.53 (s, 3H), 0.24 (br s, 6H); ESI-MS:m/z=686.9 [M+1]⁺; ¹⁹F NMR (376 MHz, D₂O) −200.77-−202.57 (m, 1F); ³¹PNMR (162 MHz, D₂O) 99.68-84.67 (m, 1P).

Step 6: Preparation of Compound 11 Sodium Salt and Compound 12 SodiumSalt

Compound 11 sodium salt. Dowex 50W×8, 200-400 (H form, 50 g) was addedto a beaker (for 45 mg of cpd 11) and washed with deionized water (2×),then added to the resin (15% H₂SO₄ in deionized water, 50 mL). Themixture was stirred for 15 min and decanted (1×). The resin wastransferred to a column with 15% H₂SO₄ in deionized water and washedwith 15% H₂SO₄ (at least 4 column volumes), and then with deionizedwater until the resin was neutral. The resin was transferred back intothe beaker, and a NaOH solution (15% NaOH in water solution, 50 mL) wasadded. The mixture was stirred for 15 min and decanted (1×). The resinwas transferred to the column and washed with 15% NaOH in water (atleast 4 column volumes) and then with water until it was neutral (atleast 4 column volumes). Compound 11 was dissolved in deionized water(50 mg in 40 mL), added to the top of the column and eluted withdeionized water. Compound 11 was eluted from the column in earlyfractions as detected by TLC (UV). The product was lyophilized to givecompound 11 sodium salt (24.3 mg, 0.033 mmol) as a white solid. ESI-MS:m/z=686.9 [M+1]⁺; ¹H NMR (400 MHz, D₂O) δ7.81 (2 H, d, J=3.6 Hz), 7.71(1 H, s), 5.99 (1 H, d, J=15.6 Hz), 5.61 (1 H, d, J=8.2 Hz), 5.16-5.32(1 H, m), 4.65-4.84 (2 H, m), 4.19-4.28 (2 H, m), 3.96-4.11 (2 H, m),3.88(1 H, d, J=4.2 Hz), 3.69-3.80 (2 H, m), 3.31 (3 H, s), −1.07-0.45 (6H, m); ¹⁹F NMR (377 MHz, D₂O) −202.06 (1 F, s); ³¹P NMR (162 MHz, D₂O)94.39 (1 P, br s).

Compound 12 sodium salt. Dowex 50W×8, 200-400 (H form, 50 g) was addedto a beaker (for 50 mg of cpd 12) and washed with deionized water (2×)then added to the resin (15% H₂SO₄ in deionized water, 50 mL). Themixture was stirred for 15 min and decanted (1×). The resin wastransferred to a column with 15% H₂SO₄ in deionized water and washedwith 15% H₂SO₄ (at least 4 column volumes), and then with deionizedwater until the resin was neutral. The resin was transferred back intothe beaker, and a NaOH solution (15% NaOH in water solution, 50 mL) wasadded. The mixture was stirred for 15 min and decanted (1×). The resinwas transferred to the column and washed with 15% NaOH in water (atleast 4 column volumes) and then with water until it was neutral (atleast 4 column volumes). Compound 12 was dissolved in deionized water(50 mg in 40 mL), added to the top of the column and eluted withdeionized water. Compound 12 eluted from the column in early fractionsas detected by TLC (UV). The product was lyophilized to give compound 12sodium salt (43.3 mg, 0.059 mmol) as a white solid. ESI-MS: m/z=686.9[M+1]⁺; ¹H NMR (400 MHz, D₂O) δ8.11 (s, 1 H), 8.06 (s, 1H), 7.94 (s,1H), 6.28 (d, J=16.06 Hz, 1H), 5.89 (d, J=8.53 Hz, 1H), 5.44-5.60 (m,1H), 5.27 (td, J=8.97, 4.39 Hz, 1H), 4.86-5.00 (m, 1H), 4.46-4.55 (m,2H), 4.37 (br d, J=11.80 Hz, 1H), 4.19-4.28 (m, 1H), 4.19-4.28 (m, 1H),4.15 (br dd, J=11.67, 2.13 Hz, 1H), 4.02 (br d, J=12.30 Hz, 1H), 3.54(s, 3H), −0.01-0.75 (m, 6H); ¹⁹F NMR (377 MHz, D₂O) −201.59 (s, 1 F);³¹P NMR (162 MHz, D₂O) 94.01 (br s, 1 P).

Example 9

Step 1: Preparation of Compound 8a

Compound 7b (10.46 g, 7.241 mmol) was dissolved in anhydrous CH₂Cl₂ (100mL) and 4 Å molecular sieves powder (4.0 g) was added to this solution.The resulting heterogeneous mixture was degassed under reduced pressureand purged with Argon several times. After stirring this mixture at 25°C. for 30 min, borane dimethyl sulfide complex solution (2.0 M in THF,BH₃-DMS, 11.948 mL, 23.897 mmol) was added very slowly for 5 min at 0°C. After stirring the reaction for 20 min at 25° C., the reactionmixture was quickly filtered, diluted with CH₂Cl₂ (120 mL) and quenchedwith water (20 mL). The organic phase was successively washed with water(50 mL) and brine (50 mL). The aqueous layers were back-extracted withEtOAc (50 mL). The combined organic layers were concentrated underreduced pressure and the resulting crude material was purified by flashcolumn chromatography on silica gel (CH₂Cl₂/MeOH=100/1 to 10/1) to givecompound 8a (11.1 g, 7.612 mmol) as a yellow solid.

Step 2: Preparation of Compound 8b

Compound 8a (11.1 g, 7.612 mmol) was dissolved in 80% aqueousAcOH/CH₃CN/Et₃SiH (3/1/1, v/v, 200 mL). After stirring the mixture for16 h at 37° C., the reaction mixture was diluted with EtOAc (500 mL) andthen neutralized with saturated aqueous NaHCO₃. The organic layer wassuccessively washed with water (500 mL) and brine (2×250 mL), then driedover Na₂SO₄, filtered, and the filtrate evaporated to dryness to give aresidue. The residue was purified by flash column chromatography onsilica gel (CH₂Cl₂/MeOH=100/1 to 10/1) to give compound 8b (3.6 g, 4.218mmol) as a white foam. ESI-MS: m/z 854.2 [M+H]⁺; ¹⁹F NMR (376 MHz,CD₃CN) δ−203.09 (s, 1F), −203.36 (s, 1F); ³¹P NMR (162 MHz, CD₃CN)δ116.95-116.34 (m, 1P).

Step 3: Preparation of Compound 8c

Compound 8b (1.5 g, 1.757 mmol) was co-evaporated with a mixture ofanhydrous toluene/CH₃CN (1/1, v/v, 3×20 mL) to give a white solid. Thesolid was then dissolved in anhydrous CH₃CN (80 mL) and 4 Å molecularsieves powder (2.0 g) was added to this solution. After stirring thismixture at 25° C. for 30 min, a solution of tetrazole in CH₃CN (0.45 M,31.242 mL, 14.059 mmol) was added to the solution at 25° C. The reactionmixture was stirred for 20 min at 25° C.2-Cyanoethoxybis-(N,N-diisopropylamino) phosphine (794.519 mg, 2.636mmol) was added to the solution over 20 min (in five portions). Afterstirring for 60 min, another portion of solution of tetrazole in CH₃CN(0.45 M, 10 mL) was added to this solution. After stirring for another 1h, the reaction mixture was diluted with EtOAc (100 mL) and filtered.The organic layer was washed with saturated aqueous NaHCO₃ (50 mL),brine (50 mL), dried over MgSO₄ (after stirring for 5 min followed byfiltration), and the filtrate evaporated to dryness to give compound 8c(1.76 g, crude) as a white solid. The resulting solid was used directlywithout further purification for the next step. Step 3 was repeated asecond time using the same scale.

Step 4: Preparation of Compounds 9a and 9b

To a solution of compound 8c (1.76 g, 1.848 mmol) in MeCN (30 mL) wasadded 3H-benzo[c][1,2]dithiol-3-one 1,1-dioxide (1.85 g, 9.238 mmol) at25° C. After stirring at 25° C. for 1 h, the reaction mixture wasfiltered, and the resulting cake was washed with CH₂Cl₂/MeOH (10/1, 20mL×3). The combined filtrates were concentrated under pressure to give aresidue. The residue was purified by flash column chromatography onsilica gel (CH₂Cl₂/MeOH=100/1 to 10/1) to give compound 9a (652 mg) as ayellow foam and compound 9b (660 mg) as a yellow foam.

Compound 9a was re-purified by reverse phase preparative HPLC (column:Phenomenex Gemini C18 250×50 10 μm; mobile phase: water (10 mMNH₄HCO₃)-ACN, Begin B:30; End B: 60; Flow Rate: 25 mL/min Gradient Time:15 min) to give compound 9a (225 mg, 0.229 mmol) as a white solid.Compound 9b was re-purified by reverse phase preparative HPLC (column:Waters Xbridge 150×25 5 μm; mobile phase: water (10 mM NH₄HCO₃)-ACN,Begin B:37; End B: 67; Flow Rate: 25 mL/min Gradient Time: 8 min) togive 9b (351 mg, 0.356 mmol, 19.294% yield) as a white solid. ESI-MS:m/z 985.5 [M+H]⁺.

Step 5: Preparation of Compound 13, Ammonium Salt

Compound 9a (100 mg, 0.102 mmol) was treated with MeNH₂ (33% in EtOH, 5mL) and stirred at 25° C. for 12 h. The reaction mixture wasconcentrated under reduced pressure to give a residue. The residue waspurified by reverse phase preparative HPLC (column: Agela Durashell C18150×25 5 μm; mobile phase: water (10 mM NH₄HCO₃)-ACN, Begin B: 0, End B:15%, flow rate: 35 mL/min, Gradient Time: 10 min) to give compound 13ammonium salt (56 mg, 0.08 mmol) as a white solid. ESI-MS: m/z 704.8[M+H]⁺. ¹H NMR (400 MHz, D₂O) δ8.30 (br, s, 1H), 7.93 (br s, 2H),6.41(br, d, J=15.8 Hz, 1H), 5.98-5.68 (m, 2H), 5.57 (br, s, 1H),5.24-4.95 (m, 1H), 4.62-4.35 (m, 3H), 4.29-4.12 (m, 3H), 4.02 (br d,J=9.3 Hz, 1H), 3.60 (s, 3H), -0.48 (br, s, 3H); ¹⁹F NMR (376 MHz, D₂O)−199.64-−201.37 (m, 1F); ³¹P NMR (162 MHz, D₂O) 91.37 (s, 1P), 91.31 (s,1P), 90.52 (s, 1P), 89.94 (s, 1P), 52.36 (s, 1P), 52.26 (s, 1P).

Step 6: Preparation of Compound 13 Sodium Salt

Dowex 50W×8, 200-400 (H form, 50 g) was added to a beaker (for 56 mg ofcpd 13) and washed with deionized water (2×). then added to the resin(15% H₂SO₄ in deionized water, 50 mL). The mixture was stirred for 15min and decanted (1×). The resin was transferred to a column with 15%H₂SO₄ in deionized water and washed with 15% H₂SO₄ (at least 4 columnvolumes), and then with deionized water until the resin was neutral. Theresin was transferred back into the beaker, and a NaOH solution (15%NaOH in water solution, 50 mL) was added. The mixture was stirred for 15min and decanted (1×). The resin was transferred to the column andwashed with 15% NaOH in water (at least 4 column volumes) and then withwater until it was neutral (at least 4 column volumes). Compound 12 wasdissolved in deionized water (50 mg in 40 mL), added to the top of thecolumn and eluted with deionized water. Compound 12 eluted from thecolumn in early fractions as detected by TLC (UV). Compound 13 wasdissolved in deionized water (56 mg in 30 mL), added to the top of thecolumn, and eluted with deionized water. A product eluted from thecolumn in early fractions as detected by TLC (UV). The product waslyophilized to afford compound 13 sodium salt (45.4 mg, 0.064 mmol) as awhite solid. ¹H NMR (400 MHz, D₂O) δ8.25 (s, 1H), 8.21 (s, 1H), 7.95 (s,1H), 6.42 (d, J=14.8 Hz, 1H), 5.91-5.84 (m, 1.5H), 5.76 (d, J=3.8 Hz,0.5H), 5.60-5.51 (m, 1H), 5.19-5.04 (m, 1H), 4.61-4.53 (m, 2H),4.52-4.44 (m, 1H), 4.27-4.18 (m, 3H), 4.07 (dd, J=4.0, 12.0 Hz, 1H),3.60 (s, 3H), 0.47-−0.89 (m, 3H); ¹⁹F NMR (377 MHz, D₂O) −201.93 (s,1F); ³¹P NMR (162 MHz, D₂O) δ92.47 (br dd, J=26.4, 73.4 Hz, 1P), 91.98(s, 1P), 91.71 (s, 1P), 91.24 (s, 1P), 91.19 (s, 1P), 91.10 (s, 1P),90.97 (s, 1P), 52.78 (s, 1P), 52.64 (s, 1P); ESI-MS: m/z 704.8 [M+H]⁺.

Step 7: Preparation of Compound 9c

To a solution of compound 9b (311 mg, 0.316 mmol) in MeCN/EtOH(1/1, 5mL) was added tert-butylamine (5 mL). After stirring for 2 h, thereaction mixture was concentrated under reduced pressure to give aresidue. The residue was purified by reverse phase preparative HPLC(column: Waters Xbridge 150×25 5 μm; mobile phase: water (10 mMNH₄HCO₃)-ACN, Begin B: 8; End B: 38; Flow Rate: 25 mL/min Gradient Time:8 min) to give compound 9c (243 mg, 0.277 mmol) as a white solid.

Step 8: Preparation of Compound 14 Ammonium Salt

Compound 9c (243 mg, 0.277 mmol) was treated with MeNH₂ (33% in EtOH, 10mL) and stirred at 25° C. for 12 h. The reaction mixture was thenconcentrated under reduced pressure to give a residue. The residue waspurified by reverse phase preparative HPLC (column: Agela Durashell C18150×25 5 μm; mobile phase: water (10 mM NH₄HCO₃)-ACN, Begin B: 0, End B:15%, flow rate: 35 mL/min, Gradient Time: 10 min) to afford compound 14ammonium salt (125 mg, 0.177 mmol) as a white solid. ¹H NMR (400 MHz,D₂O) δ8.23 (d, J=2.5 Hz, 2H), 8.01 (s, 1H), 6.43 (d, J=15.1 Hz, 1H),5.95-5.76 (m, 2H), 5.56 (dt, J=4.3, 8.9 Hz, 1H), 5.21-5.05 (m, 1H),4.64-4.46 (m, 3H), 4.33-4.15 (m, 4H), 3.56 (s, 3H), 0.96-−0.39 (m, 3H);¹⁹F NMR (377 MHz, D₂O) δ−201.77 (s, 1F); ³¹P NMR (162 MHz, D₂O) δ93.50(s, 1P), 92.44 (s, 1P), 52.51 (s, 1P); ESI-MS: m/z 704.8 [M+H]⁺.

Step 9: Preparation of Compound 14 Sodium Salt

Dowex 50W×8, 200-400 (H form, 50 g) was added to a beaker (for 125 mg ofcpd 14) and washed with deionized water (2×) then added to the resin(15% H₂SO₄ in deionized water, 50 mL). The mixture was stirred for 15min and decanted (1×). The resin was transferred to a column with 15%H₂SO₄ in deionized water and washed with 15% H₂SO₄ (at least 4 columnvolumes), and then with deionized water until the resin was neutral. Theresin was transferred back into the beaker, and a NaOH solution (15%NaOH in water solution, 50 mL) was added. The mixture was stirred for 15min and decanted (1×). The resin was transferred to the column andwashed with 15% NaOH in water (at least 4 column volumes) and then withwater until it was neutral (at least 4 column volumes). Compound 14 wasdissolved in deionized water (125 mg in 40 mL), added to the top of thecolumn, and eluted with deionized water. Product was eluted out in earlyfractions as detected by TLC (UV). The product was lyophilized to affordcompound 14 sodium salt (105.4 mg, 0.141 mmol) as a white solid. ¹H NMR(400 MHz, D₂O) δ8.21 (br, d, J=9.0 Hz, 2H), 7.89 (s, 1H), 6.38 (d,J=15.3 Hz, 1H), 5.89-5.72 (m, 2H), 5.67 (br, s, 1H), 5.19-5.02 (m, 1H),4.62-4.42 (m, 3H), 4.27-4.17 (m, 3H), 4.07 (br d, J=9.8 Hz, 1H), 3.57(s, 3H), 0.36 (br, s, 3H); ¹⁹F NMR (377 MHz, D₂O) δ−201.25 (s, 1F); ³¹PNMR (162 MHz, D₂O) δ92.42-90.93 (m, 1P), 52.35 (s, 1P); ESI-MS: m/z704.8 [M+H]⁺.

Example 10

Step 1: Preparation of Compound 7b

To a solution of compound 1f (5.0 g, 7.47 mmol) in acetonitrile (180 mL)was added 1H-tetrazole (0.45 M, 132.7 mL) at 25° C. After stirring thesolution for 10 min at 25° C., a solution of compound 7a (6.87g, 7.84mmol) in acetonitrile (20 mL) was added dropwise. After stirring for 2hours at 25° C., the solution was used into the next step without anyfurther purification.

Step 2: Preparation of Compound 10a

To the previous solution of compound 7b (332.7 mL, 7.44 mmol) inacetonitrile was added tert-butylhydroperoxide (3.35 g, 37.22 mmol) at25° C. After stirring at 25° C. for 1.5 h, the solution was diluted withEA (100 mL) and washed with aqueous saturated NaHCO₃ (2×100 mL) andbrine (2×100 mL). The organic layer was successively dried overanhydrous Na₂SO₄, filtered and the solvent evaporated under reducedpressure to give compound 10a (10 g) as a white solid.

Step 3: Preparation of Compound 10b

To a solution of compound 10a (10 g, crude) in acetonitrile (50 mL) wasadded triethylsilane (40 mL) and 80% acetic acid in acetonitrile (200mL) at 25° C. After stirring the solution at 50° C. for 12 hours, themixture was neutralized with aqueous saturated NaHCO₃ to pH 8. Themixture was diluted with EtOAc (500 mL) and the organic layer wassuccessively washed with aqueous saturated NaHCO₃ (100 mL), brine (100mL) and evaporated under reduced pressure to dryness. The aqueous layerwas extracted with EtOAc (2×200 mL) and evaporated under reducedpressure to dryness. The combined crude material was purified by flashcolumn chromatography on silica gel (0-9% MeOH in CH₂Cl₂, v/v) to givecompound 10b (5 g) as a white solid. ESI-MS: m/z 856.2 [M+H]⁺.

Step 4: Preparation of Compound 10c

To a solution of compound 10b (1.5 g, 1.4 mmol) in tetrahydrofuran (2mL) and acetonitrile (75 mL) was added 1H-tetrazole (0.45 M, 15.58 mL,7.01 mmol) at 25° C. It was then added a solution of3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (845.34 mg, 2.8mmol) in acetonitrile (5 mL) at 25° C. After stirring for 1.5 h at 25°C., the solution was washed with aqueous saturated NaHCO₃ (50 mL), brine(50 mL) and evaporated under reduced pressure to dryness to givecompound 10c (1.8 g) as a yellow solid which was used into the next stepwithout any further purification. ESI-MS: m/z 955.5 [M+H]⁺.

Preparation of Compound 15

Step 5: Preparation of Compounds 10d+10e

To a solution of compound 10c (1.5 g, 1.57 mmol) in DCM (100 mL) wasadded Borane dimethyl sulfide (2.36 mL, 4.71 mmol) at 0° C. for 2 mins.After stirring the mixture at 25° C. for 15 min, water (30 mL) wasadded. The resulting solution was filtered and the filtrate concentratedunder reduced pressure to give a yellow solid. The solid was dilutedwith DCM (100 mL) and the organic layer was successively washed withwater (2×100 mL), brine (3×100 mL) and concentrated under pressure togive a residue. The crude solid was purified by flash columnchromatography on silica gel (0-9% MeOH in CH₂Cl₂, v/v) to give amixture of compounds 10d and 10e (500 mg, crude) as a yellow solid.ESI-MS: m/z 969.3 [M+H]⁺.

Step 6: Preparation of Compound 15

A solution of compounds 10d and 10e (500 mg, crude) in a mixture ofethanol (14 mL) and NH₄OH (42 mL) was stirred at 50° C. for 12 hours.The solution was concentrated under pressure to give a yellow solid. Theyellow solid was purified by reverse phase preparative HPLC (Column:Synergi Polar-RP 100×30 504; Condition: water (10 mM NH₄HCO₃)-ACN; BeginB: 0, End B: 20; Gradient Time (min): 12; Flow Rate (ml/min): 25) toafford compound 15 (110 mg) as a white solid. Compound 15 was purified asecond time by reverse phase preparative HPLC (Column: PhenomenexKinetex XB-C18 150 mm×30 mm, 504; Condition: water (10 mM NH₄HCO₃)-ACN;Begin B: 0, End B: 5; Gradient Time(min): 7; Flow Rate (ml/min): 30) togive compound 15 ammonium salt (45 mg) as a white solid. ¹H NMR (400MHz, D₂O) δ8.31 (br, s, 1H), 8.15 (s, 1H), 7.94 (br, s, 1H), 6.43 (br,d, J=16.3 Hz, 1H), 5.98 (br, d, J=7.7 Hz, 1H), 5.67-5.47 (m, 1H), 5.28(br, s, 1H), 4.93 (br, s, 1H), 4.61-4.49 (m, 2H), 4.43 (br, d, J=9.7 Hz,1H), 4.26 (br, s, 2H), 4.18 (br, s, 1H), 4.05 (br s, 1H), 3.60 (s, 3H),0.33 (br s, 3H). ¹⁹F NMR (376 MHz, D₂O) δ−201.82 (s, 1F). ³¹P NMR (162MHz, D₂O) δ96.09 (br, s, 1P), −2.40 (s, 1P). ESI-MS: m/z 688.9 [M+H]⁺.

Step 7: Preparation of Compound 15 Sodium Salt

Dowex 50W×8, 200-400 (H form, 5 mL) was added to a beaker (for 45 mg ofcpd 15 ammonium salt) and washed with deionized water (2×) then added tothe resin (15% H₂SO₄ in deionized water, 50 mL). The mixture was stirredfor 15 min and decanted (1×). The resin was transferred to a column with15% H₂SO₄ in deionized water and washed with 15% H₂SO₄ (at least 4column volumes), and then with deionized water until the resin wasneutral. The resin was transferred back into the beaker, and a NaOHsolution (15% NaOH in water solution, 50 mL) was added. The mixture wasstirred for 15 min and decanted (1×). The resin was transferred to thecolumn and washed with 15% NaOH in water (at least 4 column volumes) andthen with water until it was neutral (at least 4 column volumes).Compound 15 ammonium salt was dissolved in deionized water (45 mg in 5mL), added to the top of the column, and eluted with deionized water.Product was eluted out in early fractions as detected by TLC (UV). Theproduct was lyophilized to afford compound 15 sodium salt P1 (42.6 mg)as a white solid. ¹H NMR (400 MHz, D₂O) δ8.21 (s, 1H), 8.09 (s, 1H),7.97 (s, 1H), 6.39 (br, d, J=16.1 Hz, 1H), 6.00 (br, d, J=8.3 Hz, 1H),5.70-5.52 (m, 1H), 5.23 (dt, J=4.5, 8.3 Hz, 1H), 5.04-4.88 (m, 1H),4.63-4.51 (m, 3H), 4.44 (br, d, J=11.8 Hz, 1H), 4.31-4.19 (m, 3H), 4.06(br, d, J=10.8 Hz, 1H), 3.59 (s, 3H), 0.67-0.12 (m, 3H). ¹⁹F NMR (376MHz, D₂O) δ−201.80 (s, 1F). ³¹P NMR (162 MHz, D₂O) δ95.45 (s, 1P), −2.26(s, 1P). ESI-MS: m/z 688.8 [M+H]⁺.

Preparation of Compound 15 and Compound 16

Step 5: Preparation of Compounds 10d+10e

To a solution of compound 10c (2.3 g, 2.41 mmol) in DCM (100 mL) wasadded Borane dimethyl sulfide (3.61 mL, 7.23 mmol) at 0° C. for 2 mins.After stirring the mixture at 25° C. for 15 min, water (20 mL) wasadded. The resulting solution was filtered and the filtrate concentratedunder reduced pressure to give a yellow solid. The solid was dilutedwith DCM (100 mL) and the organic layer was successively washed withwater (2×100 mL), brine (3×100 mL) and concentrated under pressure togive a yellow residue. The crude solid was purified by reverse phasepreparative HPLC (Column: Agela Durashell C18 150×25 5 μM; Condition:water (10 mM NH₄HCO₃)-ACN; Begin B: 25, End B: 55; Gradient Time (min):12; Flow Rate(ml/min): 25) to give a mixture of compounds 10d and 10e(65 mg,) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ8.81-8.71 (m, 1H),8.50 (d, J=6.2 Hz, 1H), 8.08 (br, d, J=8.4 Hz, 2H), 7.69-7.54 (m, 3H),6.59-6.50 (m, 1H), 6.37-6.26 (m, 2H), 6.02-5.86 (m, 1H), 5.26-5.04 (m,2H), 4.77-4.68 (m, 1H), 4.63-4.54 (m, 3H), 4.43 (br, d, J=6.2 Hz, 2H),4.34-4.27 (m, 2H), 3.86-3.69 (m, 2H), 2.98-2.90 (m, 3H), 2.71 (br, dd,J=5.8, 13.1 Hz, 1H), 2.61-2.49 (m, 1H), 1.22 (td, J=3.3, 6.7 Hz, 7H).ESI-MS: m/z 969.3 [M+H]⁺.

Step 6: Preparation of Compound 15 and Compound 16

A solution of compounds 10d and 10e (65 mg, crude) in a mixture ofethanol (4 mL) and NH₄OH (12 mL) was stirred at 50° C. for 12 hours. Thesolution was concentrated under pressure to give a residue. The residuewas purified by reverse phase preparative HPLC (Column: Synergi Polar-RP100×30 5 μM; Condition: water (10 mM NH₄HCO₃)-ACN; Begin B: 0, End B:20; Gradient Time (min): 12; Flow Rate (ml/min): 25) to afford compound15 (37 mg) and compound 16 (17 mg) as white solids.

Compound 15 ammonium salt: ¹H NMR (400 MHz, D₂O) δ8.41 (br, s, 1H), 8.23(br, s, 1H), 7.98 (br, s, 1H), 6.46 (br, d, J=16.3 Hz, 1H), 5.99 (br, s,1H), 5.74-5.46 (m, 1H), 5.27 (br, s, 1H), 5.07-4.91 (m, 1H), 4.61-4.50(m, 2H), 4.42 (br, s, 1H), 4.26 (br, s, 2H), 4.16 (br, s, 1H), 4.05 (br,s, 1H), 3.61 (s, 3H), 0.71-0.07 (m, 2H). ¹⁹F NMR (376 MHz, D₂O) δ−75.63(s, 1F). ³¹P NMR (162 MHz, D₂O) δ94.56 (s, 1P), −3.69 (s, 1P). ESI-MS:m/z 688.9 [M+H]⁺

Compound 16 ammonium salt: ¹H NMR (400 MHz, D₂O) δ8.37 (br s, 1H), 8.24(br s, 1H), 7.85 (s, 1H), 6.42 (d, J=14.1 Hz, 1H), 5.91-5.88 (m, 1H),5.8 (br s, 1H), 5.51 (br s, 1H), 5.38 (br s, 1H), 5.32-5.20 (m, 1H),4.56-4.48 (m, 2H), 4.45-4.36 (m, 1H), 4.28-4.17 (m, 3H), 4.09 (br d,J=11.0 Hz, 1H), 3.59 (s, 3H), 0.61-0.10 (m, 3H). ¹⁹F NMR (376 MHz, D₂O)δ−202.9 (s, 1F). ³¹P NMR (162 MHz, D₂O) δ96.67 (m, 1P), −3.02 (s, 1P).ESI-MS: m/z 689.2 [M+H]⁺.

Step 7: Preparation of Compound 15 Sodium Salt

Dowex 50W×8, 200-400 (H form, 5 mL) was added to a beaker (for 37 mg ofcpd 15 ammonium salt) and washed with deionized water (2×) then added tothe resin (15% H₂SO₄ in deionized water, 50 mL). The mixture was stirredfor 15 min and decanted (1×). The resin was transferred to a column with15% H₂SO₄ in deionized water and washed with 15% H₂SO₄ (at least 4column volumes), and then with deionized water until the resin wasneutral. The resin was transferred back into the beaker, and a NaOHsolution (15% NaOH in water solution, 50 mL) was added. The mixture wasstirred for 15 min and decanted (1×). The resin was transferred to thecolumn and washed with 15% NaOH in water (at least 4 column volumes) andthen with water until it was neutral (at least 4 column volumes).Compound 15 ammonium salt was dissolved in deionized water (37 mg in 5mL), added to the top of the column, and eluted with deionized water.Product was eluted out in early fractions as detected by TLC (UV). Theproduct was lyophilized to afford compound 15 sodium salt P1 (28.4 mg)as a white solid. ¹H NMR (400 MHz, D₂O) δ8.25 (s, 1H), 8.12 (s, 1H),7.99 (s, 1H), 6.42 (br, d, J=15.8 Hz, 1H), 6.02 (br, d, J=8.3 Hz, 1H),5.70-5.52 (m, 1H), 5.30-5.21 (m, 1H), 5.06-4.91 (m, 1H), 4.62-4.54 (m,2H), 4.44 (br, d, J=12.8 Hz, 1H), 4.30 (br, d, J=4.3 Hz, 2H), 4.21 (br,d, J=12.0 Hz, 1H), 4.06 (br, d, J=11.8 Hz, 1H), 3.60 (s, 3H), 0.71-0.09(m, 3H). ¹⁹F NMR (376 MHz, D₂O) δ−75.62 (s, 1F), −201.89 (s, 1F). ³¹PNMR (162 MHz, D₂O) δ96.08 (s, 1P), -2.24 (s, 1P). ESI-MS: m/z 688.8[M+H]⁺.

Example 11

Step 1: Preparation of (11a)

To a solution of 8b (100 mg, 0.11 mmol) in CH₃CN/THF (1:1, v/v, 4.4 mL)was added 4A molecular sieves (1 g) and 1H-tetrazole in CH₃CN (1.94 mL,0.9 mmol). After stirring the mixture at 25° C. for 0.5 h, 2-cyanoethylN,N,N′,N′-tetraisopropylphospho-rodiamidite (49.56 mg,0.16 mmol) inCH₃CN was added to the mixture. The mixture was stirred at 25° C. for 2h and 1H-tetrazole in CH₃CN (0.49 mL, 0.22 mmol, 0.45 M) was added tothe mixture. After stirring the mixture at 25° C. for 0.5 h, a solutionof 0.5 M of I₂ in THF:Py:H₂O (8:1:1; V/V/V) (0.66 mL, 0.33 mmol) wasadded to the reaction. After stirring the mixture at 25° C. for 2 h, asaturated aqueous solution of sodium thiosulfate (2 mL) was added; theresulting mixture was filtered and the filtrate was concentrated underreduced pressure to dryness. The residue was purified by reverse phasepreparative HPLC (Agela Durashell C18 150×25 5 μM; Condition: water (10mM NH₄HCO₃)—CAN A: water (10 mM NH₄HCO₃) B: MeCN; Begin B: 25% to B:55%, Gradient Time (min) 12; 100% B Hold Time (min) 2.2; FlowRate(ml/min) 25). The pure fractions were collected, solventconcentrated under reduced pressure and aqueous layer was lyophilized todryness to give 11a as a white solid (20 mg). ESI-MS: m/z 969.3 [M+1]+.

Step 2: Preparation of 17 and 18

To a solution of 11a (20 mg 0.017 mmol) in EtOH (1.5 mL) was added NH₃.H₂O (1.5 mL, 25%). After stirring the solution at 50° C. for 3 d, thereaction mixture was filtered and the filtrate was concentrated underreduced pressure to dryness. The residue was purified by reverse phasepreparative HPLC (Column: Syneri Polar-RP 100×30 5 μM Condition: water(10 mM NH₄HCO₃)-MeCN A: water (10 mM NH₄HCO₃) B: MeCN; Begin B: 0% to B:20%, Gradient Time (min) 12; 100% B Hold Time (min) 2.2; FlowRate(ml/min) 25). The pure fractions were collected and the solvent wasevaporated under reduced pressure to afford 17 (25 mg) and 18 (20 mg) aswhite solid.

Analogue 17 ammonium salt: ESI-MS: m/z 688.8 [M+1]+. ¹H NMR (400 MHz,D₂O) δ8.26 (br s, 1 H) 8.18 (s, 1 H) 7.77 (br s, 1 H) 6.38 (br d,J=14.31 Hz, 1 H) 5.77 (br d, J=8.03 Hz, 1 H)5.64(br s, 1 H) 5.30-5.52(m, 1 H) 4.95-5.13 (m, 1 H) 4.50 (br d, J=9.03 Hz, 1 H) 4.34-4.44 (m, 2H) 4.07-4.25 (m, 3 H) 3.99 (br d, J=11.04 Hz,1 H) 3.52 (s,3 H)−0.92-0.05 (m, 3 H).

Analogue 18 ammonium salt: ESI-MS: m/z 688.8 [M+1]+. ¹HNMR (400 MHz,D₂O) δ8.29 (s, 1 H) 8.18 (s, 1 H) 7.75 (s, 1 H) 6.35 (d, J=14.05 Hz, 1H) 5.76 (s, 2 H) 5.30-5.52 (m, 1 H) 4.99-5.20 (m, 1 H) 4.35-4.50 (m, 3H) 4.09-4.22 (m, 3 H) 3.94-4.03 (m, 1 H) 3.47 (s, 3 H) 0.05 (s, 3 H).

Step 3: Preparation of 17 Sodium Salt

Dowex 50W×8, 200-400 (H form, 25 g) was added to a beaker (for 33 mg ofcpd 17) and washed with deionized water (2×10 mL) then added to theresin 15% H₂SO₄ in deionized water (80 mL). The mixture was stirred for15 min and decanted (1×10 mL). The resin was transferred to a columnwith 15% H₂SO₄ in deionized water and washed with 15% H₂SO₄ (at least 4column volumes), and then with deionized water until the resin wasneutral. The resin was transferred back into the beaker, and a NaOHsolution (15% NaOH in water solution, 50 mL) was added. The mixture wasstirred for 15 min and decanted (1×10 mL). The resin was transferred tothe column and washed with 15% NaOH in water (at least 4 column volumes)and then with water until it was neutral (at least 4 column volumes).Compound 17 ammonium salt was dissolved in deionized water (33 mg in 5mL), added to the top of the column, and eluted with deionized water.Product was eluted out in early fractions as detected by TLC (UV). Theproduct was lyophilized to afford compound 17 sodium salt (12.4 mg) as awhite solid. ESI-MS: m/z=688.8 [M+1]⁺. ¹H NMR (400 MHz, D₂O) □ 8.08-8.05(m, 1H), 7.97 (s, 1H), 7.37-7.36 (m, 1H), 6.41-6.33 (m, 1H), 5.88 (d,J=8.0 Hz, 1H), 5.64 (d, J=4.4 Hz, 1H), 5.44-5.27 (m, 2H), 4.48 (d, J=2.4Hz, 1H), 4.38-4.30 (m, 2H), 4.20-4.11 (m, 2H), 3.50 (s, 3H), 3.46 (d,J=13.6 Hz, 1H), 3.22-3.18 (m, 1H); ¹⁹F NMR (376 MHz, D₂O) δ−196.87 (s,1F); ³¹P NMR (162 MHz, D₂O) δ7.80 (s, 1P), −1.22 (s, 1P).

Step 4: Preparation of 18 Sodium Salt

Dowex 50W×8, 200-400 (H form, 15 g) was added to a beaker (for 30 mg ofcpd 18) and washed with deionized water (2×10 mL) then added to theresin 15% H₂SO₄ in deionized water (80 mL). The mixture was stirred for15 min and decanted (1×10 mL). The resin was transferred to a columnwith 15% H₂SO₄ in deionized water and washed with 15% H₂SO₄ (at least 4column volumes), and then with deionized water until the resin wasneutral. The resin was transferred back into the beaker, and a NaOHsolution (15% NaOH in water solution, 50 mL) was added. The mixture wasstirred for 15 min and decanted (1×10 mL). The resin was transferred tothe column and washed with 15% NaOH in water (at least 4 column volumes)and then with water until it was neutral (at least 4 column volumes).Compound 18 ammonium salt was dissolved in deionized water (30 mg in 5mL), added to the top of the column, and eluted with deionized water.Product was eluted out in early fractions as detected by TLC (UV). Theproduct was lyophilized to afford compound 18 sodium salt (13.2 mg) as awhite solid. ESI-MS: m/z=688.8 [M+1]⁺; ¹H NMR (400 MHz, D₂O) δ8.22 (s, 1H) 8.14 (s, 1 H) 7.76 (s, 1 H) 6.34 (d, J=14.05 Hz, 1 H) 5.77 (d, J=8.28Hz, 1 H) 5.60-5.69 (m, 1 H) 5.30-5.48 (m, 1 H) 4.94-5.11 (m, 1 H) 4.49(br d, J=9.29 Hz, 1 H) 4.35-4.45 (m, 2 H) 4.17-4.23 (m, 1 H) 4.12-4.17(m, 1 H) 4.09 (br d, J=4.52 Hz, 1 H) 3.99 (br dd, J=12.05, 4.52 Hz, 1 H)3.52 (s, 3 H) −0.87-0.01 (m, 3 H). ³¹P NMR (162 MHz, D₂O) δ92.38 (br s,1 P) −1.31 (s, 1 P). ¹⁹F NMR (376 MHz, D₂O) δ−75.64 (s, 1 F) −202.91 (brs, 1 F).

By the method of Example 3, substituting appropriate reagents, thefollowing compounds may be prepared by one of skill in the art:

Cpd No. Structure 19

20

21

22

23

BIOLOGICAL EXAMPLES In Vitro Assays Biological Example 1

STING SPA Binding Assay

The human STING SPA binding assay measures displacement of tritiumlabeled 2′, 3′cGAMP (cyclic(guanosine-(2′→5′)-monophosphate-adenosine-(3′→5′)-monophosphate) tobiotinylated STING protein. A soluble version of recombinant STING wasexpressed in E.coli that lacks the four transmembrane domains andcontains residues 139-379 of Q86WV6 with an R at position 232 (H232R).Based on the allele frequency of 58% of the population, H232R isconsidered to be wild type (Yi, et. al., “Single NucleotidePolymorphisms of Human STING can affect innate immune response to cyclicdinucleotides” PLOS ONE. 2013, 8(10), e77846). The STING construct hasan N-terminal HIS tag, followed by a TEV protease cleavage site and anAVI tag to allow directed biotinylation by BirA biotin ligase (Beckettet al., A minimal peptide substrate in biotin holoenzymesynthetase-catalyzed biotinylation. (1999) Protein Science 8, 921-929).The HIS tag is cleaved after purification and prior to biotinylation.

The assay was run in 1536-well plates in a total volume of 8 μL per wellby adding 8 nM [³H]-2′3′-cGAMP and 40 nM biotin-STING protein in assaybuffer [25 mM HEPES (Corning 25-060-C1) pH 7.5, 150 mM NaCl (SigmaS5150), 0.5 mg/mL BSA (Gibco 15260-037), 0.001% Tween-20 (Sigma P7949),molecular grade water (Corning 46-000-CM)]. Test compounds (80 nL) wereadded with an acoustic dispenser (EDC Biosystems) in 100% DMSO for afinal assay concentration of 1% DMSO. Plates were centrifuged for 1 minand incubated for 60 min at room temperature. Finally, (2 μL)polystyrene streptavidin SPA beads (PerkinElmer RPNQ0306) were added andplates were sealed and centrifuged for 1 min at room temperature. Plateswere dark adapted for 2 h and read on a ViewLux (Perkin Elmer) for 12min per plate. A saturation binding curve for [³H]-2′3′-cGAMP showed aK_(D) of 3.6±0.3 nM for binding to STING, comparable to reported valuesfor the natural ligand (Zhang et al., Cyclic GMP-AMP containing mixedphosphodiester linkages is an endogenous high-affinity ligand for STING.

Other natural ligands including cyclic-di-GMP also returned values inthis assay within the expected range. Reference compound is cGAMP andresults are reported as percent inhibition and ICso values. Binding tomouse STING used a construct similar to the one described abovecontaining residues 138-378 of Q3TBT3.

Full Length Human STING Binding Assay

Human STING from residues 1-379 of Q86WV6 with an Rat position 232(H232R) with an N-terminal 6HIS tag followed by a FLAG tag, a TEVprotease cleavage site and an AVI tag for biotinylation wasrecombinantly expressed in HEK293-EXPI cells. Purified membranes wereprepared from these cells and STING expression was confirmed andquantified by immunoblot. STING containing membranes were combined withtest compound in a Greiner 384-well assay plate and incubated at roomtemperature for one hour in the same assay buffer used for the STING SPAbinding assay. Next, [³H]-2′3′-cGAMP was added and plates were incubatedfor 30 min at room temperature. Reactions were transferred to aprewashed Pall 5073 filter plate and each well was washed 3 times with50 μL assay buffer. Filter plates were dried at 50 □C for 1 h. To eachwell, 10 μL of Microscint scintillation fluid was added and plates weresealed and read on a TopCount (Perkin Elmer) for 1 min per well.

STING SPR Binding Assay

Compounds were analyzed on an 5200 biacore SPR instrument (GEHealthcare). E.coli produced truncated STING protein was immobilized ona series S streptavidin chip via biotin capture (GE Healthcare#BR100531) with. Compounds were screened at 1:2 dilutions from 100 uM to0.195 uM in run buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% P20, 1mM TECEP). Steady state affinity and kinetic evaluations were carriedout using 1:1 binding model (STING was treated as a dimer). Runparameters were as follows: 60 sec on, 300 sec off for the IFMcompounds, cyclic-di-GMP (60 sec on/60 sec off), thiol isomer 1 (60 secon/300 sec off) and cGAMP (60 sec on/1200 sec off) with a flow rate of50 μL/min and data collection at 40 Hz at 25 □C.

STING Human Cell Reporter Assay

Agonism of the human STING pathway is assessed in THP1-ISG cells(Invivogen, cat #thp-isg) derived from human THP1 monocyte cell line bystable integration of an interferon regulatory factor (IRF)-inducibleSEAP reporter construct. THP1-Blue ISG cells express a secretedembryonic alkaline phosphatase (SEAP) reporter gene under the control ofan ISG54 minimal promoter in conjunction with five interferon(IFN)-stimulated response elements. As a result, THP1-Blue ISG cellsallow the monitoring of IRF activation by determining the activity ofSEAP. The levels of IRF-induced SEAP in the cell culture supernatant arereadily assessed with alkaline phosphatase detection medium, a SEAPdetection reagent. These cells are resistant to Zeocin. 2′3′cGAMP wasused as a positive control in this assay. To run the assay, 60,000 cellswere dispensed in 30 μL/well of a white, opaque bottom tissue culturetreated 384-well plate.

Test compounds were added in a volume of 10 μL (1% DMSO finalconcentration). Compounds are initially prepared in 100% DMSO, spottedon an intermediate dilution plate and then diluted in media prior totransfer. The assay was incubated for 24 h at 37 □C, 5% CO₂ then plateswere centrifuged at 1200 rpm (120× g) for 5 min. After final incubation,90 μL of alkaline phosphatase detection medium-substrate was added toeach well of a new 384-well clear plate and 10 μL of the cellsupernatant was transferred from the assay plate to the new alkalinephosphatase detection medium-plate using a Biomek FX and mixed 4 times.Plates were incubated at RT for 20 min then absorbance at 655 nm wasdetermined on the Tecan Safire2.

STING Mouse Cell Reporter Assay

Agonism of the mouse STING pathway is assessed in RAW Lucia cells(Invivogen,cat #rawl-isg) derived from mouse RAW-264.7 macrophage cellline by stable integration of an interferon-inducible Lucia luciferasereporter construct. RAW Lucia cells express a secreted luciferasereporter gene under the control of an ISG54 minimal promoter inconjunction with five interferon (IFN)-stimulated response elements. Asa result, RAW Lucia cells allow the monitoring of IRF activation bydetermining the activity of luciferase. The levels of IRF-inducedluciferase in the cell culture supernatant are readily assessed withQUANTI-Luc™, a luciferase detection reagent. These cells are resistantto Zeocin. 2′3′cGAMP is used as a positive control in this assay. To runthe assay, 100,000 cells were dispensed in 90 μL/well of a clear, flatbottom tissue culture treated 96-well plate. Test compounds were addedin a volume of 10 μL. The assay was incubated for 24 and 48 hours at 37°C., 5% CO₂. After incubation, 20 μL of the cell supernatant from theassay plate was transferred to a new 96-well white plate and 50 uL ofQUANTI-Luc substrate was added. The plate was incubated, shaking, at RTfor 5 minutes then luminescence was read on an EnVision 2104 with 0.1 sintegration time.

Human Interferon-β Induction Assay

THP1-Blue ISG cells are used to measure the secretion of IFN-β into theculture supernatant following STING pathway activation. To run theassay, anti-IFN-β capture antibodies were coated on 96 well MultiArrayplates (Mesoscale Discovery). After a one hour incubation, plates werewashed and 50 μL supernatant from the STING human cell reporter assayplates or IFN-β standards were mixed with 20 μL Sulfotag-conjugateddetection antibody in the coated plates. Plates were incubated, shakingfor 2 h, washed, and read buffer was applied. Electrochemiluminescencewas measured on the Sectorlmager.

STING Cell Signaling Pathway Assessment

Agonism of the STING pathway was measured in THP1 BLUE ISG cells bywestern blot of phospho-STING(S366), phospho-TBK1(S172) andphospho-IRF3(S396). Briefly, 5 million cells in 90 μL nucleofectionbuffer were mixed with 10 μL test compounds. These mixtures wereelectroporated using program V-001 on an Amaxa Nucleofector (Lonza).Cells were transferred into 12 well plates with fresh media and allowedto recover for one hour at 37 □C, 5% CO₂. Cells were then washed in coldHBSS and lysed in RIPA buffer. Samples were total protein normalized andeither diluted in ProteinSimple sample buffer or LDS loading buffer.Samples were heat denatured at 95□C for 5 min, then PeggySue(ProteinSimple) was used to measure phospho- and total STING and IRF3while the NuPAGE (Invitrogen) system was used to measure TBK1. Data wasanalyzed using Compass or Licor Odyssey software, respectively.

STING In Vivo Activity

For all studies, female Balb/c mice were obtained from Charles RiverLabs (Wilmington, Mass.) and used when they were 6-8 weeks of age andweighed approximately 20 g. All animals were allowed to acclimate andrecover from any shipping-related stress for a minimum of 5 days priorto experimental use. Reverse osmosis chlorinated water and irradiatedfood (Laboratory Autoclavable Rodent Diet 5010, Lab Diet) were providedad libitum, and the animals were maintained on a 12 h light and darkcycle. Cages and bedding were autoclaved before use and changed weekly.All experiments were carried out in accordance with The Guide for theCare and Use of Laboratory Animals and were approved by theInstitutional Animal Care and Use Committee of Janssen R & D, SpringHouse, Pa. Each experimental group contained 8 mice. In vivo efficacy ina mouse CT26 tumor model was determined by implanting 500,000 CT26 coloncarcinoma tumor cells subcutaneously into Balb/c mice and allowingtumors to establish to 100-300 mm³. Compounds were injectedintratumorally formulated in phosphate buffered saline in a volume of0.1 ml per injection. Mice were administered 0.05 mg every three daysfor a total of three doses. Efficacy was measured as the percent tumorgrowth inhibition (TGI) calculated by the reduction in size of theTreated tumor volume (T) over the Control tumor volume (C) according tothe following formula: ((C−T)/(C))*100 when all control animals werestill on study. Cures were defined as the number of animals with nomeasurable tumor detected 10 tumor volume doubling times (TVDT) afterthe last dose was administered.

The resultant data are presented in Table 2.

TABLE 2 human SPR hSTING cell human human SPA reporter STING ThermoFluorIFN-β In vivo In vivo Cpd IC50 EC50 KD KD (ranking activity activity No.(μM)* (μM)* (μM) (μM) value) (% TGI) (cures) 1 >100 >100 >100 >83.33 NDND ND 2 <0.01 0.064 0.003 0.020 2205 87.1 2/8 3 >88.25 >10 ND >66.67 NDND ND 4 <0.01 0.09 0.002 0.001 2247 93.7 6/8 5 <0.01 0.16 0.008 0.0842737 93.3 7/8 6 0.023 0.12 0.017 0.310  27 73.9 1/8 7 0.06 1.12 0.0491.270 2260 94.3 5/8 8 0.035 0.11 0.042 0.510 2054 95.3 4/8 9 <0.01 0.640.000195 ND 2240 89.8 ND 10 0.012 0.54 0.00158 ND 1321 11 <0.01 0.493840 12 <0.01 0.22 3860 13 <0.01 0.59 2900 14 <0.01 0.45 4964 15 1.10817 2.88 ND—not done, human IFN-β ranking value determined by Rankingvalue determined by total cumulative IFN-β induction over the dose rangetested (0.78 to 50 uM) in THP-1 cells. *IC₅₀ and EC₅₀ are means of atleast three values.

Biological Example 2

STING Primary Human PBMC Cytokine Induction Assay

Agonism of the human STING pathway is assessed in primary humanperipheral blood mononuclear cells (PBMC) derived from human wholeblood. 1 pint (approximately 420 ml) of fresh donor blood (All CellsInc., Alameda, Calif.) is layered over Lymphocyte Separation Medium(1.077-1.080 g/ml, Corning, Manassas, Va.), then centrifuged at 500g for20 min at RT without applying break. The PBMC collected at the interfacebetween serum and Lymphocyte Separation Medium are harvested, washed,then counted. PBMC are composed of subtypes of lymphocytes andmonocytes, such as B cells, T cells, etc., and these subtypes have beencharacterized in the literature to express different levels of the STINGprotein. In response to STING agonists, such as 2′3′-cGAMP, these cellsbecome activated and are induced to express a variety of proinflammatoryand antiviral cytokines. Also, upon stimulation with STING agonists,these cells upregulate activation markers. The levels of cytokineinduction can be measured by a variety of methods including ELISA,Luminex and MSD. The levels of activation marker upregulation can bemeasured by flow cytometry.

To run the assay, 1,000,000 cells were dispensed into 225 μL/well offlat-bottom, tissue culture treated, 96-well plates. Test compounds wereadded in a volume of 25 μL at 10× concentration. Some compounds weresolubilized in 100% DMSO and the final concentration of DMSO in thecultures receiving these compounds was 1%. The assay was incubated for48 h at 37° C., 5% CO₂. 200 μl of supernatants were harvested withoutdisturbing cells on the bottom of the plate, then frozen at −20° C.until time of Luminex measurement. Luminex assays were performed usingG-CSF, IFNα2, IFNγ, IL-1b, IL-6, IL-10, IL-12 (p40), IL-12 (p′70), TNFafrom MILLIPLEX MAP Human Cytokine/Chemokine Magnetic BeadPanel-Immunology Multiplex Assay kit and IFNβ1 analyte from MILLIPLEXMAP Human Cytokine/Chemokine Magnetic Bead Panel IV kit (EMD Millipore,Billerica, Mass.), following the manufacturer's protocol. Cytokineinduction was measured using a Luminex FlexMAP 3D® instrument (LuminexCorporation, Radnor, Pa.). Analysis of collected Luminex data wasperformed using MILLIPLEX Analyst software (EMD Millipore).

Suppression of HBV Virus in PHH Cells Using Conditioned Media from STINGActivated Primary Human PBMC

Primary human hepatocytes can be infected with hepatitis B virus andduring an established infection, will produce viral proteins such asHBsAg and HBeAg that can be detected by ELISA. Therapeutic treatmentwith compounds such as entecavir can suppress HBV reproduction, whichcan be measured by decreased viral protein production. (# of cells)4×10⁵ cells/well primary human hepatocytes (BioReclamation, Westbury,N.Y.) were dispensed into 500 μL/well of flat-bottom, tissue culturetreated, 24-well plates. 24 h later, cells were infected with 30-75 moiof HBV. On the next day, the PHH were washed 3× and fresh maintenancemedia was added to the cells. Concurrently, PBMC were isolated asdescribed previously. To stimulate the PBMC, 10,000,000 cells weredispensed into 400 μL/well of flat-bottom, tissue culture treated,24-well plates. Test compounds were added in a volume of 100 μL, thenthe cultures were incubated for 48 h at 37° C., 5% CO₂. Supernatantswere harvested. Cells were measured for activation marker upregulationusing flow cytometery. Briefly, cells were stained with fluorescentlylabeled antibodies directed to CD56, CD19, CD3, CD8a, CD14, CD69, CD54,CD161, CD4 and CD80. Samples were analyzed on an Attune NxT flowcytometer (Thermo Fisher, Carlsbad, Calif.)

From the stimulated PBMC cultures, a portion of supernatant was reservedfor cytokine detection by Luminex, as described previously. The rest ofthe supernatant was divided in half, and one aliquot was stored at 4° C.for use on d8 of the assay. The other aliquot of supernatant was diluted1:1 with 2X PHH media, then added to the d4 infected PHH cells. After 96h, the spent media was changed and supernatant was added at a dilutionof 1:1 with 2X PHH media. At this point an interim measurement of HBsAgwas performed using an HBsAg ELISA kit (Wantai Bio-pharm, Beijing,China). Following 96 h, the media was collected and HBsAg was measured.

TABLE 3 Table 3: Fold induction of cytokines in PBMC cultures stimulatedwith CDN compounds. Cpd No. IL-6 IL-10 IFN-γ IL-1b IFN-α TNFa IL-12p40IL-12p70 G-CSF IFN-β 1 1.1 2.9 401.3 0.6 1.3 26.1 5.2 0.0 0.1 0.0 2 0.31.8 133.2 0.1 1.4 5.1 1.4 nt 0.0 nt 2 7.1 42.7 19.1 11.9 6.3 11.5 1.425.4  0.8 13.6  3 0.6 2.0 370.4 0.2 2.6 10.9 0.5 nt 0.0 nt 3 4.8 39.80.7 3.5 0.2 0.9 4.0 1.5 3.6 0.2 4 0.0 0.4 0.1 0.0 0.1 0.1 0.7 nt 0.0 nt5 1.4 2.9 605.1 0.9 1.4 50.4 2.1 nt 0.1 nt 7 1.0 6.4 502.1 0.8 38.0 29.50.5 420.5  0.0 62.2  8 0.6 1.2 133.7 0.3 6.5 12.8 0.5 nt 0.0 nt2′3′-cGAMP 2.5 5.9 17.4 1.4 6.8 5.6 1.0 5.5 0.4 16.0  PBS 1.0 1.0 1.01.0 1.0 1.0 1.0 1.0 1.0 1.0 DMSO 0.0 1.2 0.2 0.0 0.1 0.1 0.8 nt 0.0 ntFold induction is calculated by measuring the concentrations of thecytokine induced after 48 h by approximately 20 μM of compound, thendividing by base line levels of cytokine production of cells incubatedwith PBS. The data is the average of multiple donors over threeexperiments. nt = not tested.

TABLE 4 Table 4: Fold induction of cytokines in PBMC cultures stimulatedwith higher concentrations of CDN compounds. Top Conc Cpd No. (μM) IL-6IL-10 IFNγ IL-1β IFNα2 TNFα IL12p40 IL12p70 G-CSF IFNβ1 1 111.1 0.7 0.42.0 1.7 0.9 3.4 0.7 35.2 1.1 nt 2 40 2523.6 61.0 3225.5 544.8 27.0 643.49.6 252.7 227.0 18.2 3 40 491.4 21.2 4.0 102.8 1.4 7.2 3.1 3.7 7.4  0.44 111.1 0.1 0.0 1.0 0.1 0.3 0.6 0.0 7.0 0.0 nt 5 111.1 0.2 0.0 3.1 2.20.5 4.4 0.1 34.5 0.3 nt 7 111.1 321.9 4.1 2088.8 113.2 1033.1 244.6 0.627.7 2.6 14.1 8 111.1 0.4 0.0 2.1 0.7 0.6 1.4 0.0 39.4 0.1 nt 9 405084.7 121.4 4776.7 5072.5 106.4 1003.1 26.6 640.0 775.6 22.1 10 402791.9 37.7 3104.9 342.6 41.1 555.4 25.5 274.0 58.4 20.7 14 40 2209.324.1 4760.1 288.8 44.7 811.7 22.9 574.2 295.5 18.7 13 40 2536.1 50.06065.9 445.5 39.5 881.8 32.0 686.4 246.2 16.6 2′3′-cGAMP 40 454.0 12.11919.1 251.2 27.8 117.1 1.8 17.1 14.1 13.5 DMSO 0.5 0.3 0.4 0.6 0.5 0.40.5 0.6 1.2 nt Fold induction is calculated by measuring theconcentrations of the cytokine induced after 48 h the indicatedconcentration of compound, then dividing by base line levels of cytokineproduction of cells incubated with PBS. The data is the average ofmultiple donors over three experiments. nt = not tested.

TABLE 5 Conditioned media from PBMCs stimulated with CDN can suppressviral load of HBV infected PHH cells. PBMCs were stimulated with theindicated CDN at 20, 4, 0.8 μM for 48 h. Supernatants were mixed withfresh media at a ratio of 1:1, then added to HBV infected PHH cells.HBsAg production was measured 8 days later. The data is an average oftwo independent donors. Cpd No. EC50 (μM) 2 8.24E−04 3 88119.3 78.51E−05

TABLE 6 CDN activate PBMC. PBMCs were stimulated with 20 μM of CDN for48 h. Cells were assessed by flow cytometry for upregulation of CD54 onmonocytes. The fold increase in Mean Fluoresence Intensity wascalculated relative to the levels on resting cells. The data is anaverage of two independent donors. Cpd No. MEI 2 5.0 3 2.0 7 5.12′3′-cGAMP 4.5 PBS 1.0

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purposes of illustration, itwill be understood that the practice of the invention encompasses all ofthe usual variations, adaptations and/or modifications as come withinthe scope of the following claims and their equivalents.

The invention claimed is:
 1. A compound of Formula (I):

wherein: R_(1A) is hydroxy or fluoro and R_(1c) is hydrogen; R_(1B) isselected from the group consisting of hydroxy, thiol, and BH₃; B₁ isselected from the group consisting of rings b1 and b2;

R_(2A) is selected from the group consisting of hydroxy and methoxy;R_(2B) is selected from the group consisting of hydroxy, thiol, and BH₃;provided that one or both of R_(1B) and R_(2B) are BH₃ and neither ofR_(1A)and R_(2A) are OH; or an enantiomer, diastereomer, orpharmaceutically acceptable salt form thereof.
 2. The compound of claim1 wherein B₁ is b2


3. The compound of claim 1 that is:

or a pharmaceutically acceptable salt from thereof.
 4. A pharmaceuticalcomposition comprising a compound of any one of claims 1 to 3 and atleast one of a pharmaceutically acceptable carrier, a pharmaceuticallyacceptable excipient, and a pharmaceutically acceptable diluent.
 5. Thepharmaceutical composition of claim 4, wherein the composition is asolid oral dosage form.
 6. The pharmaceutical composition of claim 4,wherein the composition is a syrup, an elixir or a suspension.
 7. Apharmaceutical composition comprising a compound of claim 3 and at leastone of a pharmaceutically acceptable carrier, a pharmaceuticallyacceptable excipient, and a pharmaceutically acceptable diluent.
 8. Amethod of treating a disease, syndrome, or condition modulated by STING,comprising administering to a subject in need thereof a therapeuticallyeffective amount of the compound of claim
 1. 9. A method of treating adisease, syndrome, or condition, wherein said disease, syndrome, orcondition is affected by the agonism of STING, comprising administeringto a subject in need thereof a therapeutically effective amount of thecompound of claim
 1. 10. The method of claim 9 wherein said disease,syndrome, or condition is cancer.
 11. The method of claim 10 whereinsaid cancer is selected from the group consisting of melanoma, coloncancer, breast cancer, prostate cancer, lung cancer, and fibrosarcoma.12. The method of claim 9, wherein said disease, syndrome, or conditionis viral infection.
 13. The method of claim 11, wherein the viralinfection is hepatitis B.
 14. A method of treating a disease, syndrome,or condition selected from the group consisting of viral infection,melanoma, colon cancer, breast cancer, prostate cancer, lung cancer, andfibrosarcoma, comprising administering to a subject in need thereof atherapeutically effective amount of the composition of claim
 4. 15. Themethod of claim 9, comprising administering to a subject in need thereofa therapeutically effective amount of the compound of claim
 3. 16. Acompound of Formula (I):

wherein: R_(1A) and R_(1c) is CH₂ such that R_(1A) and R_(1c) are takentogether with the atoms to which they are attached to form a 5-memberedring; R_(1B) is selected from the group consisting of hydroxy, thiol,and BH₃; B₁ is selected from the group consisting of rings bi and b2:

R_(2A) is selected from the group consisting of hydroxy and methoxy;R_(2B) is selected from the group consisting of hydroxy, thiol, and BH₃;provided that one or both of R_(1B) and R_(2B) are BH₃, or anenantiomer, diastereomer, or pharmaceutically acceptable salt formthereof.
 17. The compound of claim 16 that is:

or a pharmaceutically acceptable salt from thereof.
 18. The compound ofclaim 1 that is:

or a pharmaceutically acceptable salt from thereof.
 19. The compound ofclaim 1 that is:

or a pharmaceutically acceptable salt from thereof.